Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections

ABSTRACT

The present invention provides nucleic acid molecules containing nucleotide sequences encoding helminth aminopeptidase enzymes, and antigenic fragments and functionally-equivalent variants thereof, their use in the preparation of vaccines, for use against helminth parasites, and synthetic polypeptides encoded by them.

This application is a divisional of U.S. Ser. No. 09/129,366, filed Aug.5, 1998 which is a continuation of U.S. Ser. No. 08/335,844, now U.S.Pat. No. 6,066,503, issued May 23, 2000 which was a filing under 35 USC§ 371 of PCT/GB93/00943, filed May 7, 1993 which claimed priority fromGB 9209993.6, filed May 8, 1992.

The present invention relates to the preparation of protective antigensby recombinant DNA technology for use as anthelmintic agents and asprotective immunogens in the control of diseases caused by helminthparasites.

Helminth parasites are responsible for a wide range of diseases andinfestations of domestic animals which, leading as they do to loss ofproduction and even animal mortality, are of considerable economicimportance. Thus for example, the blood feeding nematode Haemonchusinfects the lining of the gastrointestinal tract of ruminants, causinganaemia and weight loss and if untreated frequently leads to death.Animals infected with the related non-blood feeding nematode Ostertagiasimilarly fail to thrive and may die if untreated. Other genera ofhelminths of economic importance include Trichostrongylus andNematodirus which cause enteritis in various animals, and trematodes.

Problems are also caused by nematodes such as hookworms (eg. Necator,Ancylostoma Uncinaria and Bunostomum ssp) and flukes (eg. Fasciola,Paramphistomum and Dicrocoelium) and their relatives which in additionto ruminants and domestic pets, also infect humans, frequently withfatal results.

Control of helminth parasites presently relies primarily on the use ofanthelmintic drugs combined with pasture management. Such techniqueshave a number of drawbacks however—frequent administration of drugs andpasture management are often not practical, and drug-resistant helminthstrains are becoming increasingly widespread.

There is therefore a need in this field for an effective anti-helminthvaccine and many efforts have been concentrated in this area in recentyears. However, as yet there are no commercially available molecular orsub-unit vaccines for the major helminth species, particularly for thegastrointestinal nematodes of ruminants, such as Haemonchus andOstertagia.

Most promising results to date have been obtained with novel proteinsisolated from Haemonchus, which have potential as protective antigensnot only against Haemonchus but also against a range of other helminths.In particular the protein doublet H11OD, found at the luminal surface ofthe intestine of H. contortus has been shown to confer protectiveimmunity against haemonchosis in sheep.

H11OD from H. contortus has an approximate molecular weight of 110kilodaltons (kd) under reducing and non-reducing conditions, as definedby SDS-PAGE, and is described in W088/00835 and W090/11086. The term“H11OD” as used herein refers to the protein doublet H110D as defined inW088/00835 and W090/11096. Corresponding proteins have also recentlybeen shown in other helminth species, eg. Necator americanus.

A number of methods for the purification of H11OD have been described inW088/00835 which suffice for the characterisation of the protein, andmay be scaled up to permit production of the protein in experimentallyand commercially useful quantities. There is however a need for animproved and convenient source from which to prepare not only H11OD butalso related antigenic proteins, especially for a process based onrecombinant DNA technology and expression of the proteins in suitablytransformed prokaryotic or eukaryotic organisms.

The present invention seeks to provide such an improved procedure.Sequence determination of cDNAs for H11OD from Haemonchus contortus hasbeen performed and the predicted amino acid sequences have been found todisplay homology with a family of integral membrane aminopeptidases(systematic name: α-amino acyl peptide hydrolase (microsomal)).

The mammalian integral membrane aminopeptidases are located in severaltissues, eg. on the microvillar brush border of intestines, and kidney.Their role in the kidney is unclear, but in the intestine their functionis to cleave the small peptides which are the final products ofdigestion (for reviews, see Kenny & Maroux, 1982; Kenny & Turner, 1987;Noren et al, 1986; Semenza, 1986).

In one aspect the present invention thus provides nucleic acid moleculescomprising one or more nucleotide sequences which encode helminthaminopeptidase enzymes or antigenic portions thereof substantiallycorresponding to all or a portion of the nucleotide sequences as shownin FIG. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or sequences coding forhelminth aminopeptidase enzymes which are substantially homologous withor which hybridise with any of said sequences.

A nucleic acid according to the invention may thus be single or doublestranded DNA, cDNA or RNA.

Variations in the aminopeptidase-encoding nucleotide sequences may occurbetween different strains of helminth within a species, betweendifferent stages of a helminth life cycle (e.g. between larval and adultstages), between similar strains of different geographical origin, andalso within the same helminth. Such variations are included within thescope of this invention.

“Substantially homologous” as used herein includes those sequenceshaving a sequence identity of approximately 50% or more, eg. 60% ormore, and also functionally-equivalent allelic variants and relatedsequences modified by single or multiple base substitution, additionand/or deletion. By “functionally equivalent” is meant nucleic acidsequences which encode polypeptides having aminopeptidase activitieswhich are similarly immunoreactive ie. which raise host protectiveantibodies against helminths.

Nucleic acid molecules which hybridise with the sequences shown in FIG.2, 3, 4 or 5 (composed of SEQ ID NOS: 1 to 15) or any substantiallyhomologous or functionally equivalent sequences as defined above arealso included within the scope of the invention. “Hybridisation” as usedherein defines those sequences binding under non-stringent conditions(6×SSC/50% formamide at room temperature) and washed under conditions oflow stringency (2×SSC, room temperature, more preferably 2×SCC, 42° C.)or conditions of higher stringency eg. 2×SSC, 65° C. (where SSC=0.15MNaCl, 0.015M sodium citrate, pH 7.2).

Methods for producing such derivative related sequences, for example bysite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids are well known in the art, as aremethods for determining whether the thus-modified nucleic acid hassignificant homology to the subject sequence, for example byhybridisation.

Provision of a nucleic acid molecule according to the invention thusenables recombinant aminopeptidase enzymes, or immunogenic fragmentsthereof, to be obtained in quantities heretofore unavailable, therebypermitting the development of anti-helminth vaccines.

In another aspect the present invention thus provides nucleic acidmolecules comprising one or more nucleotide sequences encoding one ormore polypeptides capable of raising protective antibodies againsthelminth parasites, which sequences incorporate one or more antigenicdeterminant-encoding regions from the aminopeptidase-encoding sequencesas shown in FIG. 2, 3, 4 or 5 (composed from SEQ ID NOS: 1 to 15).

The present invention also extends to synthetic polypeptides comprisingone or more amino acid sequences constituting an aminopeptidase enzymeor antigenic portions thereof, substantially corresponding to all or aportion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQID NOS: 1 to 15), or a functionally-equivalent variant thereof otherthan a synthetic polypeptide corresponding to the protein doublet H110D,or a synthetic polypeptide corresponding to any of the individualpolypeptide sequences disclosed in WO90/11086.

Alternatively viewed, the invention also provides synthetic polypeptidescomprising an amino acid sequence constituting an aminopeptidase enzymeor an antigenic portion thereof, substantially corresponding to all or aportion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQID NOS: 1 to 15) or a functionally-equivalent variant thereof,substantially free from other Haemonchus contortus components.

The invention further extends to vaccine compositions for stimulatingimmune responses against helminth parasites in a human or non-humananimal, comprising at least one synthetic polypeptide as defined above,together with a pharmaceutically acceptable carrier.

W090/11086 discloses a number of polypeptide or partial polypeptidesequences obtained by proteolytic digestion or chemical cleavage of theprotein doublet H110D as follows:

(a) Met Gly Tyr Pro Val Val Lys Val Glu Glu Phe (b) Met Gly Phe Pro ValLeu Thr Val Glu Ser (c) Met Gly/Phe Asn Phe Lys Ile Glu/Val Thr/Glu AlaGly (d) Met Lys Pro/Glu Thr/Val Lys Asp/Ala Thr/Lys Leu - Ile Thr (e)Met Leu Ala Leu Asp Tyr His Ser - Phe Val (f) Met Leu Ala Glu/Tyr AspGln/Ala Glu Asp Val (g) Met Gly Phe Pro Leu Val Thr Val Glu Ala Phe Tyr(h) Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala Phe/Thr Ala Thr Ser/GlyPhe Pro (i) Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/ Leu Leu Asn/Gly(j) Lys - Thr Ser Val Ala Glu Ala Phe Asn (k) Lys Ala Ala Glu Val AlaGlu Ala Phe Asp - Ile -   -   -  Lys Gly (l) Lys Ala Val Glu Val/Pro AlaGlu Ala Phe Asp Asp Ile Thr? Tyr  -   -  Gly Pro Ser (m) Lys  -  Glu GluThr Glu Ile Phe Asn Met (n) Lys  -   -   -  Pro Phe Asn/Asp Ile Glu AlaLeu (o) Asp Gln Ala Phe Ser Thr Asp Ala Lys (p) Met Gly Tyr Pro Val ValLys Val Glu Glu Phe Ala Thr Ala Leu (q) Met Gly Phe Pro Val Leu Thr ValGlu Ser - Tyr?  -  Thr (r) Met Glu/Phe Asn Phe Leu Ile Glu/Val Thr/GluAla Gly  -  Ile Thr (s) Met Gly Phe Leu Val Thr Val Glu Ala Phe Tyr -Thr Ser (t) Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala Phe/Thr Ala ThrSer/Gly Phe Pro (u) Met Lys Pro/Glu Thr/Val Leu Asp/Ala Thr/LysLeu  -  Ile Thr  -  Gly (v) Met Leu Ala Leu Asp Tyr His Ser  -  Phe ValGly? (w) Met Leu Ala Glu/Tyr Asp Gln/Ala Glu Asp Val (x) Lys His/TyrAsn/Val Ser Pro Ala Ala Glu Asn/ Leu Leu Asn/Gly (y) Lys  -  Thr Ser ValAla Glu Ala Phe Asn (z) Lys Ala Ala Glu Val Ala Glu Ala Phe Asp  -Ile  -   -   -  Lys Gly (aa) Lys Ala Val Glu Val/Pro Ala Glu Ala Phe AspAsp Ile Thr? Tyr  -   -  Gly Pro Ser (bb) Lys  -  Glu Gln Thr Glu IlePhe Asn Met (cc) Lys  -   -   -  Pro Phe Asn/Asp Ile Glu Ala Leu (dd)Asp Gln Ala Phe Ser Thr Asp Ala Lys

Uncertainties are shown either by the form Phe/Gly, where the firstthree letter code represents the most likely correct amino acid based onthe strength of the signal, or by a question mark; a sign “-” means anunknown residue.

The specific individual polypeptide sequences which are disclosed inWO09/11086 are disclaimed.

The term “polypeptide” as used herein includes both full length protein,and shorter peptide sequences.

“Functionally equivalent” as used above in relation to the polypeptideamino acid sequences defines polypeptides related to or derived from theabove-mentioned polypeptide sequences where the amino acid sequence hasbeen modified by single or multiple amino acid substitution, addition ordeletion, and also sequences where the amino acids have been chemicallymodified, including by glycosylation or deglycosylation, but whichnonetheless retain protective antigenic (immunogenic) activity. Suchfunctionally-equivalent variants may occur as natural biologicalvariations or may be prepared using known techniques, for examplefunctionally equivalent recombinant polypeptides may be prepared usingthe known techniques of site-directed mutagenesis, random mutagenesis,or enzymatic cleavage and/or ligation of amino acids.

Generally, the synthetic polypeptides according to the inventionrepresent protective antigenic sequences. The term “protective antigen”as used herein defines those antigens capable of generating ahost-protective (immunogenic) immune response ie. a response by the hostwhich leads to the generation of immune effector molecules, antibodiesor cells which sterilise the fecundity of, damage, inhibit or kill theparasite and thereby “protect” the host from clinical or sub-clinicaldisease and loss of productivity. Such a protective immune response maycommonly be manifested by the generation of antibodies which are able toinhibit the metabolic function of the parasite, leading to stunting,lack of egg production and/or death.

The synthetic polypeptides according to this aspect of the invention maybe prepared by expression in a host cell containing a recombinant DNAmolecule which comprises a nucleotide sequence as broadly describedabove operatively linked to an expression control sequence, or arecombinant DNA cloning vehicle or vector containing such a recombinantDNA molecule. Alternatively the polypeptides may be expressed by directinjection of a naked DNA molecule according to the invention into a hostcell.

The synthetic polypeptide so expressed may be a fusion polypeptidecomprising a portion displaying the immunogenicity of all or a portionof an aminopeptidase enzyme and an additional polypeptide coded for bythe DNA of the recombinant molecule fused thereto. For example, it maybe desirable to produce a fusion protein comprising a syntheticaminopeptidase or other polypeptide according to the invention coupledto a protein such as β-galactosidase, phosphatase,glutathione-S-transferase, urease, hepatitis B core antigen (Francis etal., 1989) and the like. Most fusion proteins are formed by expressionof a recombinant gene in which two coding sequences have been joinedtogether with reading frames in phase. Alternatively, polypeptides canbe linked in vitro by chemical means. All such fusion or hybridderivatives of aminopeptidase-encoding nucleic acid molecules and theirrespective amino acid sequences are encompassed by the presentinvention. Such suitable recombinant DNA and polypeptide expressiontechniques are described for example in Sambrook et al., 1989.Alternatively, the synthetic polypeptides may be produced by chemicalmeans, such as the well-known Merrifield solid phase synthesisprocedure.

Further aspects of the invention include use of a nucleic acid moleculeor a synthetic peptide or polypeptide as defined above, for thepreparation of a vaccine composition for stimulating immune responses ina human or non-human, preferably mammalian animal against helminthparasite infections.

Alternatively viewed, the invention also provides a method ofstimulating an immune response in a human or non-human, preferablymammalian, animal against a helminth parasite infection comprisingadministering to said animal a vaccine composition comprising one ormore polypeptides encoded by a nucleotide sequence as defined above.

A vaccine composition may be prepared according to the invention bymethods well known in the art of vaccine manufacture. Traditionalvaccine formulations may comprise one or more synthetic polypeptidesaccording to the invention together, where appropriate, with one or moresuitable adjuvants eg. aluminium hydroxide, saponin, QuilA, or morepurified forms thereof, muramyl dipeptide, mineral oils, or Novasomes,in the presence of one or more pharmaceutically acceptable carriers ordiluents. Suitable carriers include liquid media such as saline solutionappropriate for use as vehicles to introduce the peptides orpolypeptides into a patient. Additional components such as preservativesmay be included.

An alternative vaccine formulation may comprise a virus or host cell eg.a microorganism (eq. vaccinia virus, adenovirus, Salmonella) havinginserted therein a nucleic acid molecule (eq. a DNA molecule) accordingto this invention for stimulation of an immune response directed againstpolypeptides encoded by the inserted nucleic acid molecule.

Administration of the vaccine composition may take place by any of theconventional routes, eg. orally or parenterally such as by intramuscularinjection, optionally at intervals eg. two injections at a 7-28 dayinterval.

As mentioned above, the amino acid translation of the nucleotidesequences depicted in FIG. 2, 3, 4 or 5 show sequence homology with afamily of integral membrane aminopeptidase enzymes. This was determinedby searching various databases available in the Genetics Computer GroupSequence analysis software package, version 7.01, November 1991(Devereux et al., (1984)), using translations of the sequences shown inFIG. 2, 3, 4 or 5. Two such comparisons are shown in FIG. 6.

Expression of the aminopeptidase-encoding sequences according to theinvention can, as mentioned above, be achieved using a range of knowntechniques and expression systems, including expression in prokaryoticcells such as E. coli and in eukaryotic cells such as yeasts or thebaculovirus-insect cell system or transformed mammalian cells and intransgenic animals and plants. Particularly advantageously, thenucleotide sequences may be expressed using the transgenic nematodesystem such as the system for the nematode Caenorhabditis described forexample in Fire, (1986); Fire et al., (1989); Spieth et al., (1988); Hanet al., (1990).

A further aspect of the invention provides a method for preparing asynthetic polypeptide as defined above, which comprises culturing aeukaryotic or prokaryotic cell containing a nucleic acid molecule asdefined above, under conditions whereby said polypeptide is expressed,and recovering said polypeptide thus produced.

Further aspects of the invention thus include cloning and expressionvectors containing nucleotide sequences according to the invention. Suchexpression vectors include appropriate control sequences such as forexample translational (eg. start and stop codes) and transcriptionalcontrol elements (eg. promoter-operator regions, ribosomal bindingsites, termination stop sequences) linked in matching reading frame withthe nucleic acid molecules of the invention.

Vectors according to the invention may include plasmids and viruses(including both bacteriophage and eukaryotic viruses) according totechniques well known and documented in the art, and may be expressed ina variety of different expression systems, also well known anddocumented in the art. Suitable viral vectors include, as mentionedabove, baculovirus and also adenovirus and vaccinia viruses. Many otherviral vectors are described in the art.

A variety of techniques are known and may be used to introduce suchvectors into prokaryotic or eukaryotic cells for expression, or intogerm line or somatic cells to form transgenic animals. Suitabletransformation or transfection techniques are well described in theliterature.

Transformed or transfected eukaryotic or prokaryotic host cells ortransgenic organisms containing a nucleic acid molecule according to theinvention as defined above, form a further aspect of the invention.

Eukaryotic expression systems in general, and the nematode expressionsystem in particular, have the advantage that post-translationalprocessing, and particularly glycosylation can occur—in the case of thetransgenic nematode system, a glycosylation corresponding to that foundin the native protein may be expected. This represents an importantaspect of the invention, since in many cases post-translationalprocessing is required for the recombinant protein to express optimumbiological activity.

Mammalian cell expression systems, also have a number of advantages.Mammalian host cells provide good reproduction of the native form andprotective epitopes of the antigen since a eukaryotic expression systemwill give rise to more similar glycosylation patterns, disulphidebonding and other post-translational modifications than E. coli whichmay produce an insoluble protein requiring refolding and having poorreproduction of the native form. In addition mammalian glycosylation isunlikely to induce an immune response which distracts from a protectiveanti-protein response. For protection of humans and domestic animals, itis thus preferable to use human or animal fibroblast or myeloma celllines such as HeLa—a human cell line; BHK—baby hamster kidney cells;VERO, a monkey kidney cell line; FR3T3, Fisher rat fibroblasts; NIH3T3,a mouse fibroblast cell line; C127I, a mouse mammary tumour cell line;CV-1, African green monkey kidney fibroblasts; 3T6, mouse embryofibroblasts; L cells, a mouse cell line; CHO, a Chinese Hamster Ovarycell line; NSO NSI, SP2 and other mouse myeloma cell lines and ratmyeloma cell lines such as YB2/0 and Y3.

Vectors appropriate for different classes of mammalian cell lines arewell known in the art. In general, these will comprise a promoter and/orenhancer operably connected to a nucleotide sequence encoding theantigen or fragment thereof. Suitable promoters include SV40 early orlate promoter, eg. PSVL vector, cytomegalovirus (CMV) promoter, mousemetallothionein I promoter and mouse mammary tumour virus long terminalrepeat. The vector preferably includes a suitable marker such as a genefor dihydrofolate reductase or glutamine synthetase. Vectors of thosetypes are described in W086/05807, W087/04462, W089/01036 andW089/10404.

Transfection of the host cells may be effected using standardtechniques, for example using calcium phosphate, DEAE dextran,polybrene, protoplast fusion, liposomes, direct microinjection, genecannon or electroporation. The latter technique is preferred and methodsof transfection of mammalian cell lines using electroporation aredescribed by Andreason et al., 1980. In general, linear DNA isintroduced more readily than circular DNA.

In the case of the protein H11OD, it has been found to have a unique andunusual glycosylation pattern, which is thought to contribute toimmunoactivity since many monoclonal antibodies so far obtained to H110Dfrom Haemonchus recognise carbohydrate epitopes which may be ofimportance in developing useful vaccines.

In particular the following glycosylation pattern for H110D fromHaemonchus has been demonstrated:

-   i. about 65% of oligosaccharides are N-linked, the remainder    O-linked;-   ii. the major part (eg. about 48%) of the N-linked oligosaccharide    is of the complex class;-   iii. substantially all (eg. greater than 95%) of the    oligosaccharides are uncharged;-   iv. the relative molar content of the constituent monosaccharides is    N-acetylgalactosamine 1.0, fucose 3.6, galactose 4.1, glucose 4.4,    mannose 6.2 and N-acetylglucosamine 5.2;-   v. the oligosaccharides, other than the major oligosaccharide    (designated oligosaccharide D), are substantially resistant to    degradation by a broad range of exo-glycosidases (eg.    α-D-mannosidase, β-D-mannosidase, β-D-glucosidase,    β-D-galactosidase, α-D-galactosidase, α-L-fucosidase,    β-D-xylosidase, β-D-N-acetylqlucosaminidase).

Such oligosaccharides and glycoproteins containing them form a furtheraspect of this invention.

Oligosaccharide D of the Haemonchus H110D glycoprotein is of theN-linked type and has a novel structure consisting of two fucoseresidues attached by an α-1,3 linkage and an α-1,2 linkage to a mannose(N-acetylglucosamine) 2 core.

Another aspect of the invention thus provides an oligosaccharide havingthe structure:

and more particularly the structure:

especially when linked to a protein, eg. a recombinant protein such as ahelminth aminopeptidase-protein or an antigenic fragment thereof, orwhen used to generate anti-idiotypic antigens for immunisationespecially of very young animals.

Animal glycoproteins generally have fucose α-1,6 linkages and the fucoseα-1,3 linkage of the oligosaccharide of the present invention is anunusual feature.

This invention will now be described in more detail with particularreference to the protein H110D from Haemonchus contortus. However, by avariety of techniques such as histochemistry and DNA hybridisation,H110D equivalents have been observed in other parasite species. It isbelieved that the H110D protein is a multigene complex and that inaddition, the nucleotide sequences encoding it, may exhibit sequencevariations between different strains and different life cycle stages ofthe helminth. Moreover there may exist multiple enzyme forms(isoenzymes) which may be differentially expressed at different stages,or in different strains. In this study DNA sequences, and thus thepredicted amino acid sequences, have been determined from cDNA clonesand PCR products obtained from mRNA corresponding to the H110D gene byrecombinant DNA technology from different sources, and at differentparasitic stages of H. contortus life cycle.

Sequencing of cDNA and PCR products has enabled us to identify threeclosely related H110D sequences which are here designated H11-1 (SEQ IDNO: 19), H11-2 (SEQ ID NO: 20) and H11-3 (SEQ ID NO: 21). H11-1comprises three contiguous and overlapping sequences, cDNA clone AustB1(SEQ ID NO: 6), PCR product A-648 (SEQ ID NO: 9) and at the 3′ end PCRproduct 014-178 (SEQ ID NO: 12); H11-2 comprises the PCR products A-650and 2.5 kb (SEQ ID NOS: 10 and 7 respectively); H11-3 comprises the PCRproducts 3.5 kb and A-649 (SEQ ID NOS: 8 and 11 respectively). Thespecific relationships between the individual sequenced cDNA and PCRproduct clones and H11-1, -2 and -3 are summarised in FIG. 1 and shownin detail in FIGS. 3, 4 and 5.

Differences and variations in the sequences obtained from the cDNAclones and PCR products have been observed, as can be seen in particularfrom FIGS. 2, 3, 4 and 5 (composed of SEQ ID NOS: 1 to 15 and 19 to 21)and as summarised in Table 1.

TABLE 1 Homologies of the deduced amino acid sequences obtained bytranslation of the nucleotide sequences shown in FIG. 2. % Similarity %Identity H11-1:H11-2 77 63 H11-1:H11-3 79 65 H11-2:H11-3 82 69

The differences can be attributed to different mRNAs (of the multigenefamily). In addition, the variations may be due, at least in part, todifferent variants of the H110D-encoding sequence or mRNA present atdifferent stages of the life cycle or in strains differing ingeographical origin.

Table 2 additionally shows levels of identity and similarity between thecorresponding predicted amino acid sequences and two published mammalianaminopeptidase sequences.

TABLE 2 Homologies of the H110D amino acid sequences with rataminopeptidase M (ApM) and mouse aminopeptidase A (ApA). % Similarity %Identity H11-1:ApM 55 32 H11-1:ApA 55 31 H11-2:ApM 52 31 H11-2:ApA 54 31H11-3:ApM 53 32 H11-3:ApA 52 30

FIG. 1 shows a map of the H. contortus H110D cDNA and PCR product clonessequenced and their relationships and relative positions along the H110DmRNA;

FIG. 2 shows the H110D nucleotide sequences designated H11-3. (SEQ IDNO: 21, derived from cloned PCR products SEQ ID NOS: 8 and 11 and cDNAclone M1AUS, SEQ ID NO: 5), H11-2 (SEQ ID NO: 20, derived from clonedPCR products SEQ ID NOS: 7 and 10) and H11-1 (SEQ ID NO: 19, derivedfrom cloned PCR products SEQ ID NOS: 9 and 12 and cDNA clone AustB1, SEQID NO: 6);

FIG. 3 shows the sequence H11-3 (SEQ ID NO: 21) (shown in FIG. 2) withalignment of the cDNA clones M1 and M1AUS (SEQ ID NOS: 1 and 5);

FIG. 4 shows the sequence H11-2 (SEQ ID NO: 20, shown in FIG. 2) and thealignment of the cDNA clone B2 (SEQ ID NO: 4);

FIG. 5 shows the sequence designated H11-1 (SEQ ID NO: 19) and alignmentof the cDNA B1A and Aust B1 (SEQ ID NOS: 2 and 6 respectively);

FIG. 6 shows a) the predicted amino acid sequences (SEQ ID NOS: 22, 23and 24) derived from the DNA sequences H11-1, H11-2 and H11-3 shown inFIG. 2; bi) and ii) show the predicted amino acid sequence of H11-3compared with the published amino acid sequences of rat microsomalaminopeptidase M (Watt et al., 1989) and mouse microsomal aminopeptidaseA (Wu et al., 1990) respectively; identities are enclosed in boxes,dashes indicate spaces introduced to maximise the level of homologybetween the compared sequences. The conventional single letter code foramino acids is used. The horizontal line above the sequence indicatesthe position of the transmembrane region and the asterisks show theposition of the zinc-binding motif. Levels of similarity are shown inTables 1 and 2;

FIG. 7 Shows the alignments of amino acid sequences (designated Pep A,Pep B, Pep C, Pep D and Pep E) obtained from CNBr and Lys-C fragments ofH110D as previously described (International patent applicationWO90/11086 and as listed earlier, polypeptide sequences (a), (b), (e),(k) and (aa), respectively) and three new sequences (SEQ ID NOS: 16, 17and 18) obtained from H110D following digestion by elastase orthermolysin with the translations of a) H11-1, b) H11-2 and c) H11-3.

In a further-aspect the invention also provides nucleic acid moleculescomprising one or more nucleotide sequences which substantiallycorrespond to or which are substantially complementary to one or moresequences selected from the sequences of clones M1, B1A, B1A-3′, B2,M1AUS, AustB1, 014-015 (2.5PCR), 014-872 (3.5PCR clone 2), A-648 (5′ endof B1), A-650-(5′ end of 2.5PCR), A-649 (5′ end of 3.5PCR), 014-178 (3′end of AustB1 clone 2), 014-178 (3′ end of AustB1 clones 3 & 6), 014-872(3.5PCR clone 10) and 014-872 (3.5PCR clone 19), H11-1, H11-2 and H11-3,SEQ ID NOS: 1 to 15 and 19 to 21 respectively as shown in FIGS. 2, 3, 4,and 5 or sequences which are substantially homologous with or whichhybridise with any of the said sequences.

As mentioned above, comparison of the sequences of various of the clonesmentioned above, against computer databases of known sequences, revealssubstantial homology with the family of microsomal aminopeptidaseenzymes (EC 3.4.11.-). Enzymological activity and inhibitor studiesperformed with the H110D protein and sub-fractions thereof confirm thatthe protein is in fact microsomal aminopeptidase (α-amino acyl peptidehydrolase (microsomal)). Such studies have further shown that bothaminopeptidase A-like and aminopeptidase M-like activities areexhibited, and that each of the components of the H110D doubletindividually exhibit enzyme activity.

Studies with proteolytic digestion of H110D have also been carried out.Using the enzyme elastase, it was found that H110D may be partiallycleaved, forming two fractions, a detergent-soluble fraction (whichremained with the membrane) and a water-soluble fraction (which isdesignated H11S). H11S occurs in the form of a protein dimer which maybe reduced to two components. Interestingly, it was found that onlyaminopeptidase M-like activity is associated with the water-soluble H11Sfraction, whereas aminopeptidase A-like activity is only associated withthe detergent-soluble fraction.

The following Example provides a description of the studies leading todetermination of the sequences shown in FIGS. 1 to 7, with reference tothe following additional Figures in which:

FIG. 8 shows Western blots of integral membrane proteins present in adetergent extract of Haemonchus contortus adults probed with affinitypurified antibodies eluted from potential H110D clones; a) antigens in adetergent extract of Haemonchus recognised by antiserum to the extract;b) antibodies eluted from a strip such as that shown in a) re-testedagainst a blot of the detergent extract confirm the success of theelution step; c) antibodies as in b) which bind to clone M1 expressedprotein strongly recognise a region at 110 kd (and a relatively sharpband at about 205 kd; d) there is no antibody binding when anon-recombinant is used to adsorb the serum;

FIG. 9 shows a Northern blot of mRNA purified from 11, 15 and 23 day-oldHaemonchus contortus probed with a) cDNA clone M1 (SEQ ID NO: 1); b)cDNA clone M1 AUS (SEQ ID NO: 5); c) cDNA clone B1A (SEQ ID NOS: 2 and3); d) cDNA clone AustB1 (SEQ ID NO: 6); e) cloned PCR product 014-872(3.5-2, SEQ ID NO: 8); and f) cloned PCR product 014-015 (SEQ ID NO: 7).The numbers 11, 15 and 23 indicate the age of the Haemonchus from whichthe mRNA was obtained;

FIG. 10 shows Southern blots of Haemonchus contortus genomic DNA probedwith cDNA clones M1AUS (SEQ ID NO: 5), B1A (SEQ ID NOS: 2 and 3) andAustB1 (SEQ ID NO: 6) and PCR products 014-872 (3.5-2, SEQ ID NO: 8) and014-015 (SEQ ID NO: 7); a) blots were washed at a moderate stringency,b) blots were washed at a high stringency; for each probe, track 1contained a HindIII digest of λDNA as marker or was left blank, tracks 2and 3 contained EcoRI and HindIII digests respectively of Haemonchusgenomic DNA;

FIG. 11 shows Western blots of recombinant GST-M1 and GST-B1A fusionproteins probed with affinity purified antibodies to electrophoreticallypurified H110D (H110DE);

FIG. 12 shows Western blots of ConA H110D antigen probed with antiserato ConA H110D and to recombinant GST-M1 and GST-B1A fusion proteins;

FIG. 13 shows a) the results of analysis of H110D protein andaminopeptidase enzyme activities in fractions obtained by ion exchangechromatography of ConA H110D on a MonoQ column;

b) SDS-PAGE of the fractions shown in FIG. 13 a);

FIG. 14 shows a) the pH values at which fractions were obtained in afree-flow isoelectric focussing experiment;

b) SDS-PAGE under reducing conditions of the fractions from 14a) inwhich the lower band of the H110D doublet is found in Fraction 6 and theupper band in Fraction 16, with varying amounts of each in theintervening fractions;

c) Western blots of the fractions shown in 14b) probed with i)monoclonal antibodies designated TS 3/19.7 and ii) affinity purifiedpolyclonal anti-M1 antibodies; control antibodies gave no detectablereaction;

FIG. 15 shows a) the pH values at which fractions were obtained inanother free-flow isoelectric focussing experiment; b) SDS-PAGE underreducing conditions of fractions from 15a) used in enzyme assays, inwhich the lower band of the H110D doublet is found in Fractions 4-6 andthe upper band in Fractions 16-18 with varying amounts of each band inthe intervening fractions; c) microsomal aminopeptidase specificactivities of fractions shown in 15b);

FIG. 16 shows protection of sheep by vaccination with separated upper(U), lower (L), recombined (U+L) and intermediate doublet (D) bands fromH110D; a) parasite egg output, expressed as eggs per gram faeces, b)worm burden at post-mortem, relative to controls;

FIG. 17 shows protection of sheep by vaccination with a water-solublefragment (H11S) obtained from H110D by digestion with elastase and H11A,the residual detergent-soluble H110D. a) parasite egg output, expressedas eggs per gram faeces; b) worm burden at post-mortem, relative tocontrols (C);

FIG. 18 shows examples of the relationship between inhibition of Ai),Bi) aminopeptidase M-like and Aii), Bii) aminopeptidase A-likeactivities of H110D by antisera of individual sheep vaccinated withH110D with levels of protection measured by Ai, ii) % reduction of wormburden at post-mortem and Bi, ii) % reduction reduction of faecal eggcount; □ anti-H110D, ▪ anti-horse ferritin control;

FIG. 19 shows the histochemical localisation of aminopeptidase enzymeactivities in adult Haemonchus contortus the light micrographs ofcryo-sections of adult female Haemonchus contortus show aminopeptidaseactivity (red reaction product appears as dark band (arrowed) in theseblack and white photographs) associated only with the microvilli (mv) ofthe intestine (i). None of the other tissues (eg. cuticle (c),hypodermis (h), genital tract (gt), wall muscle (wm)) show activity. Ina) the substrate was L-leucine 4-methoxy-β-naphthylamide, in b) thesubstrate was L-glutamic acid α-(4-methoxy-β-naphthylamide);

FIG. 20 shows a map of the 3.5 PCR product (clone 2) (SEQ ID NO: 8)sub-cloned into the baculovirus expression vector pBlueBacII”;

FIG. 21 shows a Western blot of extracts from baculovirus-infectedinsect SpodoPtera frugiperda (Sf)₉ cells probed with anti-H110DNantibodies. Two cloned plaques, P3A and P4A expressed the full-lengthimmuno-positive H110D (arrowed), the controls did not.

EXAMPLE Methods

Construction of U.K. λgt11 LibrarymRNA Isolation

Adult Haemonchus contortus (0.5 gm) of UK origin snap-frozen in liquidnitrogen were ground in liquid nitrogen using a pre-chilled mortar andpestle. The RNA was extracted from the grindate with 10 volumes of 4 Mguanidine hydrochloride in 25 mM sodium citrate containing 0.5% w/vsarkosyl and 0.7% w/v 2-mercaptoethanol, followed by extraction withphenol and chloroform using the method of Chomczynski & Sacchi (1987).Messenger RNA (mRNA) was prepared from this by affinitychromatography-on oligo dT cellulose (twice) as described in Maniatis etal (1982) and the quality was assessed by in vitro translation using arabbit reticulocyte lysate kit and ³⁵S-methionine from AmershamInternational plc, according to the manufacturer's instructions.Polypeptides up to 120 kd were detected.

Complementary DNA Preparation

First strand complementary DNA (cDNA) was synthesized from 1 μg mRNAusing random priming and avian reverse transcriptase and the secondstrand was synthesized using a replacement reaction with RNase H and E.coli DNA Polymerase I followed by repair of 3′ overhangs using T4 DNAPolymerase, according to the method of Gubler & Hoffman (1983). Theyield of double-stranded (ds) cDNA was approximately 400 ng from 1 μgmRNA. The ds cDNA was examined by electrophoresis in a 1% agarose gelfollowed by autoradiography. The ds cDNA was in the size range 0.2-9.4kilobases-(Kb), with the majority being in the range 0.5-2.3 Kb.

Cloning of cDNA in λgt11

Non-size selected cDNA was used to construct a library in λgt11 usingthe Amersham cDNA cloning system (kit no. RPN 1280, AmershamInternational plc) and in vitro packaging extracts (kit no. N334,Amersham International plc) as described in the manufacturer'sinstructions, and EcoRI linker oligonucleotides (5′GGAATTCC). Theresulting library was plated on E. coli strain Y1090 in the presence ofisopropylthio-β-D-galactoside (IPTG) and 5-bromo, 4-chloro, 3-indolylβ-D-galactoside (X-gal), under which conditions recombinant λgt11 appearas clear (“white”) plaques and wild-type non-recombinant λgt11 as blueplaques. The library contained 90% white plaques and the cloningefficiency was calculated to be 4×10⁷ plaque forming units (pfu)/μg cDNAand a library titre of 2×10⁶ plaque forming units per ml. Analysis ofthe DNA from 20 recombinants picked at random revealed an average insertsize of 0.51 Kb. However this mean was distorted by one clone with aninsert of 3.5 Kb. The majority of the inserts were >300 base pairs (bp).This unamplified λgt11 library derived from UK worm mRNA was thenimmunoscreened.

Preparation of Antibody Probes Antiserum to Integral Membrane Proteins

Intestines were dissected from adult Haemonchus contortus (of UK origin)and homogenised in ice-cold phosphate buffered saline (PBS), pH 7.4,containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mMphenylmethylsulphonyl fluoride (PMSF). The homogenate was centrifugedfor 10 minutes using a microfuge and the pellet resuspended in the samebuffer containing 0.1% v/v Tween 20 (Tween is a Trade mark). Afterre-centrifugation, the pellet was resuspended in the same buffercontaining 2% v/v Triton X-100 and extracted for two hours at 4° C. Thisextract was centrifuged as above, to obtain a supernatant containingintegral membrane proteins (IMP).

A sheep was hyperimmunised with IMP in Freund's Complete Adjuvant (FCA)by intramuscular injection of 50, 50, 120 and 130 μg of IMP given onweeks 0, 7, 11 and 15. Six weeks after the final injection, serum washarvested, and designated serum EE-068.

Preparation of Integral Membrane Proteins by Detergent Extraction ofHaemonchus Contortus

An extract was prepared by homogenizing worms in 5-10 volumes of PBScontaining 1 mM EDTA and 1 mM PMSF. The suspension was centrifuged at10,000×g for 20 minutes at 4° C. and the pellet washed in the samebuffer containing 0.1% v/v Tween 20 then. extracted with 5 volumes 2%v/v Triton X-100 as described above. The supernatant was re-centrifugedat 100,000×g for 1 hour, and the resulting supernatant, which wasenriched in H110D but contained other IMP, was used in Western blottingexperiments and for the preparation of non-denatured H110D (see below).

Preparation of H110D and Affinity Purified Anti-H110DN

The extract enriched for H110D, was subjected to affinity chromatographyon ConA-agarose followed by ion exchange chromatography on MonoQ (asdescribed in WO88/00835 and WO90/11086). The purified H110D was injectedintramuscularly into lambs in FCA. Three doses of 100 μg were given at 3week intervals. Serum collected from the lambs 4 weeks after the finalinjection was affinity purified by absorption to a column containingpurified H110D which had been coupled to cyanogen bromide activatedSepharose (Pharmacia). Coupling of H110D to the Sepharose, binding ofantiserum and elution of anti-H110D antibodies were according to theinstructions supplied by Pharmacia. These affinity purified antibodiesare designated anti-H110DN. The “N” distinguishes these antibodies fromthose raised to denatured, electrophoretically purified H110D, which aredesignated anti-H110DE.

Western Blotting

Western blotting was carried out using standard procedures (Johnstone etal., 1982).

Isolation and Characterisation of Clones

Immunoscreening of the U.K. λgt11 Library

The method used to immunoscreen the library was essentially as describedby Bowtell et al (1986). Prior to use, the serum (EE-068) was depletedof anti-E. coli antibodies by absorption with lysates and whole cells ofE. coli Y1090. The library was plated on E. coli Y1090 cells at adensity of 10³ pfu per 90 mm diameter plate. Plates were overlaid withnitrocellulose filters impregnated with IPTG and incubated overnight.The filters were washed with TBST (50 mM Tris, pH 7.4, 150 mM NaCl,0.05% v/v Tween 20) and then blocked with 5% v/v horse serum in TBST for2 hours. Serum EE-068 diluted 1 in 200 in TBST containing 5% horse serumwas added and the filters incubated for 4 hours with gentle rocking. Thefilters were again washed in TBST, then incubated with horseradishperoxidase (HRP)-conjugated horse anti-sheep IgG diluted 1 in 500 inTBST containing 5% v/v horse serum for 2 hours. (Anti-serum to sheep IgGwas raised in a horse, the anti-sheep IgG purified by affinitychromatography on a sheep IgG Sepharose column, and the antibodiesconjugated to HRP by the method of Nakane & Kawaoi, 1974.) Filters werefurther washed in TBST and positive plaques detected using 0.6 mg/ml3,3′-diaminobenzidine (DAB) and 0.1% v/v hydrogen peroxide. Twenty-fiveputative positives were picked and were rescreened with affinitypurified anti-H110DN as described above. Following this secondary screenrecombinants were still positive, with the clone designated as M1 givingthe strongest signal.

Affinity Purification of Antibody on Recombinant Phage

Confluent plates were prepared on E. coli Y1090 lawns by plating 10³ pfuof each of the antibody-positive λclones or non-recombinant λgt11negative control phage. The lawns were incubated for 4 hours at 42° C.then overlaid with filters impregnated with IPTG and further incubatedovernight at 37° C. The filters were removed from the plates and washedin TBST prior to being blocked with 5% v/v horse serum for 1 hour. Thefilters were then incubated with a 1 in 100 dilution of antiserum EE-068for 6 hours, before being thoroughly rinsed with TBST. Bound antibodieswere eluted from the filters by two applications of 2 ml of elutionbuffer (5 mM glycine, 500 mM NaCl, 0.2. Tween 20, pH 2.3) for 2 to 3minutes each, neutralised by addition of 200 μl of 1 M tris-HCl, pH 7.4,diluted 1 in 200 and used to immunoscreen a Western blot of anH110D-enriched extract.

DNA Sequencing of the M1 Clone

Lambda DNA was isolated from the M1 clone according to the methodsdescribed in Maniatis et al (1982). The 2.38 Kb KpnI-SstI fragmentcontaining the 300 bp M1 fragment was isolated by gel electrophoresis,purified using a GENECLEAN kit (Stratagene) (GENECLEAN is a registeredtrade mark of BI0101) and subcloned into pBluescriptII SK⁺ (Stratagene).The EcoRI fragment was purified using the same methods and re-subclonedinto the same vector.

The nucleotide sequence of the M1 insert was determined using a T7Sequencing kit (Pharmacia, U.K.), using both the M13 forward and reverseprimers.

Preparation of Australian λgt11 and λZap cDNA LibrariesmRNA Isolation

5 gm adult Haemonchus contortus (Australian McMaster susceptible strain)snap-frozen in liquid nitrogen were ground in liquid nitrogen and theRNA extracted using hot phenol by the method of Cordingley et al.(1983). Yield of total RNA was 10.35 mg. 1.3 mg of this RNA was used toprepare mRNA by affinity chromatography on oligo dT cellulose (2sequential purifications) using the method described by Maniatis et al.(1982). Yield of mRNA was 21.6 μg. Quality of mRNA was assessed by invitro translation in rabbit reticulocyte lysate in the presence of³⁵S-methionine (Amersham) according to the supplier's instructions. Thetranslation products obtained had clearly distinguished bands includingbands >200 kd in size as demonstrated by electrophoresis onSDS-polyacrylamide gels followed by fluorography.

cDNA Synthesis and Library Preparation

1 μg mRNA was used to make cDNA by priming with oligo dT or randomprimers, using a cDNA synthesis kit from Amersham International plcfollowing the manufacturer's instructions. Yield was 115 ng doublestranded (ds) cDNA. The quality of the cDNA was examined byelectrophoresis of the ³²P-labelled DNA on an alkaline agarose gel asdescribed by the Amersham cDNA kit instructions. Size of the cDNA (bycomparison with λ-HindIII markers, New England Biolabs) was from 150 bpto >10 Kb, with most of the products being in the size range 0.6-5 Kb.The oligo dT-primed and random-primed ds cDNAs were pooled and ligatedto excess EcoRI 8-mer linkers (5′GGAATTCC3′ New England Biolabs,Catalogue No. 1018) which had been labelled with γ-³²P-ATP and T4polynucleotide kinase. The linkered cDNA was digested with EcoRI andexcess linkers were removed by Sepharose 4B (Pharmacia) chromatographyaccording to the methods described by Maniatis et al. (1982). Fractionsfrom the column were pooled in two lots, one containing cDNA larger than2 Kb and one of cDNA less than 2 Kb. Each pool was then ligatedseparately to 1 μg EcoRI cut, phosphatased λZapII arms (Stratagene) andpackaged separately using Gigapack Gold (Stratagene, registeredtrademark). The larger sized cDNA yielded 1.3×10⁵ recombinants and thesmaller cDNA 1.4×10⁵ recombinants; these were pooled to yield a libraryof 2.7×10⁵. The λZap library was amplified by plating on XL1-Blue cells(Stratagene) at 2×10⁴ pfu per 135 mm plate. The titre of the amplifiedlibrary was 7×10⁷ pfu/ml.

A further 2 μg mRNA was used to make cDNA as described above, but usingonly oligo dT as primer. The yield of ds cDNA was 740 ng. This cDNA wastreated with EcoRI methylase as described in Maniatis et al (1982) priorto addition of EcoRI linkers, and in this case 12-mer linkers(5′CCGGAATTCCGG3′ New England Biolabs, Catalogue No. 1019) were used.Following digestion of the linkered cDNA with EcoRI, all fractions froma Sepharose 4B column which contained cDNA were pooled, and ligated to 2μg EcoRI cut, phosphatased λgt11 arms (Stratagene). The ligation mix wassplit in two and packaged with two lots of Gigapack Gold (Stratagene);these were pooled to yield a λgt11 library of 7×10⁶ pfu. The library wasamplified by plating on ST9 cells at 5×10⁵ pfu per 135 mm plate. Thetitre of the amplified λgt11 library was 4.5×10¹¹ pfu/ml.

Screening of the Australian λgt11 Library with Antisera to H110D

Antisera were raised by injecting sheep with H110D protein (of UKorigin) which had been electro-eluted from polyacrylamide afterelectrophoresis in SDS according to the following method: ConA H110Dprepared as described in WO 88/00835 and WO 90/11086 was electrophoresedon SDS polyacrylamide gels (Laemmli 1970) to obtain electro-elutedH110D. After electrophoresis, the area of the polyacrylamide gelcontaining H110D was cut out, placed in an electroeluter (Atto) andelution carried out for 3 hours at 10 watts. The electroeluted H110D(designated H110DE) was concentrated on a Centriprep 10 (Amicon) andbuffer exchanged on a PD10 column (Pharmacia) into 50 mM ammoniumbicarbonate/0.07% SDS, mixed with adjuvants and then injected intosheep. Immunoglobulins from the sera were precipitated with ammoniumsulphate (Johnstone and Thorpe, 1982). The precipitated antibodies wereresuspended at 60 mg/ml in phosphate buffered saline, dialysed againstphosphate buffered saline and diluted 1:10 in Tris buffered saline (TBS)containing 5% w/v low fat milk powder. 10 mg of ConA H110D was made to0.5% SDS, heated to 100° C. for 3 minutes and dried onto anitrocellulose filter. Following washes with TBS containing 0.2% v/vTween 20 and 0.5% Triton X-100 (TBSTT) the filter was incubated for 1 to2 hours at room temperature with the antibodies to H110DE. After washingthe filter for 2 hours with TBSTT, the bound antibodies were eluted with3 ml of 0.1M glycine, 0.15M NaCl pH 2.6 for 2 minutes and immediatelyadjusted to neutral pH by the addition of 75 μl of 1.5 M Tris pH 8.0.These affinity purified antibodies, designated anti-H110DE, were used toscreen 5×10⁵ pfu of the Australian λgt11 cDNA library as describedabove.

5×10⁵ recombinants from the λgt11 library derived from AustralianHaemonchus contortus were immunoscreened and three positives picked.Following further screening two of these recombinants were stillpositive and were designated B1A and B2.

Sequencing of B1A and B2 Clones

The two clones were digested with EcoRI, yielding a single insert ofapproximately 500 bp for B1A and three fragments, B2A (about 400 bp),B2B (about 100 bp) and B2C (about 100 bp), for B2. These were subclonedinto pBluescript SK⁺ (Stratagene) and sequenced using a Sequenase 2.0kit (United States Biochemicals).

Expression of clones M1 and B1a

The M1 (SEQ ID NO: 1) and B1A (SEQ ID NOS: 2 and 3) inserts wereexpressed in E. coli, using a pGEX vector (Smith and Johnson 1988). Thisvector expresses proteins at the C-terminus of Schistosoma japonicumglutathione-S-transferase (GST). The M1 and B1A EcoRI inserts wereligated to EcoRI-cut, phosphatased pGEX1 and transformed into E. colistrain JM101 according to the methods described in Maniatis et al. 1982.Eight progeny were picked from each transformation and 2 ml cultureswere grown for 6 hours at 37° C. IPTG was added to induce fusion proteinsynthesis, and the incubation continued overnight. Cells were harvestedby centrifugation, disrupted by boiling in sample buffer (Laemmli,1974), and the extracts analysed by SDS-PAGE and by Western blottingusing affinity purified sheep antibodies specific for the SDS-denaturedH110D doublet (anti-H110DE—see above). Bound antibodies were detectedusing alkaline-phosphatase conjugated rabbit anti-sheep IgG alkalinephosphatase conjugate (Jackson Immunoresearch) followed by colourdevelopment with 5-bromo,4-chloro,3-indolyl phosphate (BCIP) andnitroblue tetrazolium (NBT). Cultures of immunopositive clones weregrown and induced as above and disrupted by sonication. The sonicateswere separated into soluble and insoluble fractions by centrifugation(Sorvall RC-2B centrifuge, HS4 rotor, 7000 rpm, 30 minutes, 4° C.). Theinsoluble pellets were resuspended in 8 M urea by sonication, andsamples of fractions examined by SDS-PAGE. The fusion proteins werefound to be in the insoluble inclusion body fraction. Each of thesepreparations was used to vaccinate 2 sheep three times at 150 μg fusionprotein per dose in Freunds adjuvants. Positive control sheep wereimmunised with native ConA H110D protein, and negative control sheepwere immunised with solubilised protein from E. coli containing the pGEXvector without an Haemonchus insert. Sera from vaccinated sheep wereanalysed by Western blotting against H110D.

Screening of the Australian λZap Library by DNA Hybridisation with M1and B1A Inserts

M1 and B1A plasmid DNAs (cloned in pBluescript) were digested with EcoRIand the inserts isolated by electrophoresis in TBE (tris-borate-EDTA; 89mM tris-borate, 89 mM boric acid, 2 mM EDTA pH approximately 8.3) bufferin 1% agarose gel, followed by purification using a GENECLEAN kit. Theisolation and purification were repeated to avoid contamination of theprobe with plasmid DNA sequences which would hybridise to λZAPsequences, causing unacceptable levels of background. The purifiedinsert DNAs were labelled with α-³²P-dCTP using a Nick Translation kitfrom Promega Biotech according to the manufacturer's instructions.Labelled DNA was separated from unincorporated label by spin columnchromatography (Maniatis et al., 1982). Eight 135 mm plates of the λZAPlibrary were plated at 10⁵ pfu/plate, and plaque lifts performed ontonitrocellulose filters (Maniatis et al., 1982). Following baking in avacuum oven for two hours at 80° C., filters were prehybridised for twohours, then hybridised at 42° C. overnight (as described below in theSouthern Blot analysis section). Four filters were screened with the M1probe and four with the B1A probe. Filters were washed twice in 2×SSCcontaining 0.5% SDS, once in 1×SSC containing 0.5% SDS and once in0.5×SSC containing 0.5% SDS, all at 50° C., and autoradiographed.Potential positive plaques were picked, and re-screened with the probes.High titre phage stocks were prepared from confirmed positives(designated M1AUS for the M1-hybridising clone and AustB1 for theB1A-hybridising clone) and the clones rescued into pBLUESCRIPT accordingto the λZAP manufacturer's instruction manual (Stratagene), using BB4 asthe host E. coli strain. Plasmid DNA minipreps of the resultant progenywere prepared by alkaline lysis (Maniatis et al., 1982) and digestedwith EcoRI. Digests were analysed by agarose gel electrophoresis.

Sequencing of the M1AUS Insert

DNA sequencing was carried out on purified pBLUESCRIPT plasmid DNA usingthe United States Biochemicals version 2.0 Sequenase kit, according tothe manufacturer's instructions. For the first sequencing reactionsprimers from the ends of the vector sequence were used to prime thereactions. The sequencing data obtained from these reactions was used todesign a second pair of primers and from the data generated with thesesecond primers a third pair were designed. In this way the DNA wassequenced by ‘walking along’ from both the 5′ and 3′ ends.

Sequencing of the AustB1 Insert

This was carried out using Sequenase 2.0 T7 polymerase (USBBiochemicals) as described for the sequencing of the M1AUS insert.

Polymerase Chain Reactions

Preparation of cDNA

mRNA (1 μg) from 11 day old post-infection U.K. H. contortus, preparedas described for adult UK worms, was mixed with T17 adaptor-primer(5′GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 3′) in diethyl pyrocarbonate(DEPC)-treated water, then heated to 65° C. for 5 minutes andimmediately placed on ice. Methylmercury hydroxide was added to a finalconcentration of 28.6 mM and the mixture incubated at room temperaturefor 3 minutes. 2-mercaptoethanol was added to a final concentration of14.2 mM and the mixture was placed on ice. To synthesize cDNA, RNAseGuard (Pharmacia) was added to 1 unit/μl, Reverse Transcriptase buffer(Life Sciences) to 1 times concentration, dATP, dGTP, dCTP and dTTP eachto 1 mM, and AMV Reverse Transcriptase (Life Sciences) to 2 units/μl(all given as final concentrations). The reaction was incubated at 41°C. for 75 minutes, then extracted with phenol and chloroform andpurified by spun column chromatography (Maniatis et al, 1982). Thepurified reaction mix was diluted 2.5-fold and stored at 4° C.

PCR Amplification of the cDNA Using M1AUS-Specific Primers

PCR reactions were carried out using a Programmable Thermal cycler (M.J.Research Inc.). The reaction mix contained 1 μl out of the 250 μldiluted cDNA prepared as described above, 25 μmol of the first strandT17-adaptor-primer, 25 μmol of second strand amplification primer(either that based on positions 865-884 (5′ACGGGTGTTCGGTTTCCGTAT 3′) orthat based on positions 30-49 (5′GCTGAATCTAACTCCAATCC 3′) of the M1AUSsequence (SEQ ID NO: 5)), 1× Taq buffer (Northumbria Biologicals Ltd)and 0.5 mM each of dATP, dTTP, dGTP and dCTP, in a 100 μl reactionvolume and covered with 40 μl mineral oil to prevent evaporation. Thismix was then heated in the thermal cycler to 95° C. for 2 minutes thenheld at 72° C. Whilst at 72° C. 2 units of Taq Polymerase (NorthumbriaBiologicals Ltd) was added and mixed gently with the other reactants.The following program was then carried out in the thermal cycler:

Step 1 Anneal at 50° C. for 5 minutesStep 2 Extend at 72° C. for 40 minutesStep 3 Denature at 94° C. for 40 secondsStep 4 Anneal at 50° C. for 2 minutesStep 5 Extend at 72° C. for 3 minutesStep 6 39 cycles of steps 3 to 5Step 7 Final extension at 72° C. for 15 minutes

Step 8 Hold at 4° C.

These conditions were established from Frohman et al., (1988).Cloning of the PCR products

The PCR products from the above reactions were separated byelectrophoresis in an agarose gel. Bands of DNA of approximately 2.5 and3.5 kb were electroeluted onto glass fibre (Whatman), phenol extractedand purified by G50 chromatography (Pharmacia) (Sambrook et al., 1989).The purified DNA was ligated into pT7Blue T-vector (Novagene)-followingthe manufacturer's instructions.

Sequencing of the 2.5 kb and 3.5 kb PCR Products

DNA sequencing was carried out with a Sequenase 2.0 kit (USBiochemicals) using the “oligonucleotide walking” technique described inthe section on sequencing of M1AUS.

Polymerase Chain Reactions for the 5′ Ends

Preparation of First Strand cDNA

1 μg of mRNA from 11 day post-infection UK Haemonchus contortus preparedas described for adult worms was mixed with a constant primer(5′AAIGAAAGCGGATGGCTTGAIGC 3′) designed from a conserved region inAustB1 and the 2.5 kbPCR and 3.5 kbPCR products (SEQ ID NOS: 6, 7 and 8respectively). The mixture was heated to 65° C. for 5 min., placed onice and methyl mercury hydroxide added to a final concentration of 28.6mM. The mixture was incubated at room temperature for 5 min., then2-mercaptoethanol added to a final concentration of 142 mM and themixture placed on ice. First strand DNA was prepared using reagents fromthe 5′ RACE system (Gibco/BRL) at a final concentration of 20 mMTris/HCl pH 8.4, 50 mM KCl, 2.5 mM MgCl₂, 100 μg/ml BSA, 0.5 mM of dATP,dCTP, dGTP, dTTP. 200 Units of Superscript Reverse Transcriptase wereadded and the reaction was incubated at 42° C. for 30 min. and thenheated at 55° C. for 5 min. RNAse H was added to a final concentrationof 100 Unit/ml and the reaction incubated at 55° C. for 10 min. and thenplaced on ice. The cDNA was purified through a Glassmax spin column(Gibco/BRL) and stored at −20° C.

C-Tailing of the cDNA

⅕ of the first strand cDNA was heated at 70° C. for 5 min then chilledon ice for 1 min. Reagents from the 5′RACE system (Gibco/BRL) were addedto a final concentration of 10 mM Tris/HCl pH 8.4, 25 mM KCl, 1.25 mMMgCl, 50 ug/ml BSA, 0.2 mM dCTP. 500 Units/ml Terminal transferase wereadded and the reaction incubated at 37° C. for 10 min, then heated at70° c. for 15 min and stored on ice.

PCR Amplification using AustB1, 2.5 kbPCR and 3.5 kbPCR Specific Primers

The PCR reactions were carried out in a programmable Thermal Cycler(M.J. Research Inc.). For the 3′ end one of 3 primers was used.

1. A primer specific for the 2.5 kb PCR product based on positions 374to 394 (5′TGTTGTGGCTAATTTCGTCCA 3′).2. A primer specific to the 3.5 kb product based on positions 1210 to1229 (5′CATCTTIAGTTATCTGACCAG 3′).3. A primer specific for the cDNA clone AustB1 based on positions 357 to377 (5′ GACCATCGCTGATGAAGTCGG 3′). For the 5′ end of the reactions acommon ‘Anchor primer’(5′CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG3′) was used. Eachreaction mixture contained 4 μl of the 50 μl of C-tailed cDNA, 25 pMolof the appropriate 2 primers, 1× Taq polymerase buffer(Boehringer/Mannheim) and 0.5 mM each of dATP, dCTP, dGTP and dTTP to afinal volume of 100 μl. This mix was covered with 50 μl of mineral oiland heated to 95° C. in the cycler for 2 min. The reaction mix was heldat 80° C. whilst 0.6 units of Taq Polymerase were added and then putthrough the following programme:

1. Anneal at 50° C. for 5 min. 2. Extend at 72° C. for 10 min. 3.Denature at 94° C. for 45 sec. 4. Anneal at 50° C. for 1 min. 5. Extendat 72° C. for 2.5 min.

6. 39 cycles of 3 to 5.

7. Extend at 72° C. for 15 min. 8. Hold at 4° C. Cloning of the 5′ PCRProducts

The PCR products were separated by electrophoresis on an agarose gel andbands of the expected size, circa 1.3 kb, were cut out, the DNA purifiedusing a GENECLEAN kit and ligated into PT7Blue T-Vector (Novagene)according to the manufacturer's instructions.

Polymerase Chain Reaction for the Production of the 3′ End of AUSTB1

The first strand cDNA used was that described for the production of cDNAfor use with M1AUS primers. A specific primer from 1414 to 1435(5′TCTTGAAGAAATGAAAAAGCTT 3′) in AustB1 (SEQ ID NO: 6) was used with theT17 Adaptor primer used for the M1AUS PCR and the reactions carried outin a thermal cycler (M.J. Research Inc). The reaction mixture consistedof 25 pMol of each primer, 2 μl of cDNA, 1× Taq Polymerase buffer(Boehringer Mannheim), 0.5 mM dATP, dCTP, dGTP and dTTP in 100 μl. Thesewere covered with 50 μl mineral oil and heated to 95° C. for 2 min. 0.6μl of Taq polymerase was added and the same programme cycle carried outas for the 5′ PCR described above.

Cloning and Sequencing of the 3′ Products.

The products of the PCR were separated by electrophoresis in an agarosegel and a band of the expected size, 1.3 kb, cut out and the DNApurified using a GENECLEAN kit and ligated into PCR-Script (Stratagene)according to the manufacturer's instructions.

Sequencing of the Cloned PCR Products

The DNA from the PCR clones was sequenced using a Sequenase 2.0 kit(United States Biochemical) as instructed by the manufacturer.Oligonucleotide primers were used to “walk along” the DNA of the clonesfrom both the 5′ and 3′ ends.

Analysis of all DNA Sequences

Sequences were analysed using the GCG (Genetics Computer Group) SequenceAnalysis Software Package, Devereux et al., 1984.

Northern and Southern Blot Analyses Preparation of Northern Blots

Northern blots were performed in formaldehyde gels essentially asdescribed in Maniatis et al. (1982). mRNA samples (from 11-, 15- and23-day old adult H. contortus) were treated with 17.5% v/v formaldehydeand 50% v/v formamide in MOPS buffer (20 mM3-(N-morpholino)propanesulphonic acid, pH 7.0, 8 mM sodium acetate, 1 mMEDTA) at 65° C. for 15 minutes, and cooled on ice. Gels wereelectrophoresed in MOPS buffer, and blotted onto Duralon membranes bycapillary transfer as described in Sambrook et al., (1989).

Preparation of Southern Blots

Two gm of adult Haemonchus contortus which had been snap-frozen inliquid nitrogen were ground to a fine powder n liquid nitrogen. Thepowder was added slowly to 25 ml of lysis buffer (0.05 M Tris-HCl, pH 8,0.1 M EDTA, 1% w/v Sarkosyl, 0.05 mg/ml proteinase K (BoehringerMannheim)) and incubated for two hours at 65° C. The suspension was thenextracted twice with one volume of phenol plus chloroform, twice withtwo volumes of chloroform, and ethanol. precipitated. The precipitatedgenomic DNA was resuspended in 20 ml of Tris, EDTA buffer (TE, pH 8)overnight at 4° C. on a rocking table, then dialysed against two changesof one litre of TE. RNA was removed by incubating with DNase-free RNaseA Type 1 (Sigma) at a final concentration of 20 μg/ml, at 37° C. for onehour, followed by one extraction with phenol-chloroform, one extractionwith chloroform, and ethanol precipitation, as above. The precipitatedgenomic DNA pellet was washed twice with 70% v/v ethanol, andresuspended in one ml TE, as above.

Genomic DNA was digested with EcoRI or HindIII (25 μg of DNA in eachdigest) overnight at 37° C., then electrophoresed at 5 μg per track on a1%. w/v agarose gel in Tris-acetate buffer. The gel was Southern blottedby capillary transfer as described in Maniatis et al., (1982) ontoHybond-N membrane (Amersham International). DNA was fixed onto themembrane using ultraviolet light, according to the manufacturer'srecommendations.

Preparation of Probes

pBLUESCRIPT plasmids containing the M1AUS, B1A or AustB1 inserts weredigested with EcoRl. pT7Blue plasmids containing 3.5 kbPCR productinserts were digested with BamHl and those containing 2.5 kbPCR productinserts were digested with BamHl and Xbal. Digests were electrophoresed,the inserts recovered and radioactively labelled with α-³²P-dCTP by nicktranslation as described above under screening of the λZAP library.

Hybridization Conditions

For Southern blots

The membranes were cut into strips and pre-hybridised in hybridisationbuffer as described earlier, for 3 hours at 28° C. Genomic DNA Southernblot strips were hybridised to each of the above probes overnight at 28°C., washed twice at room temperature (24° C.) then twice at 42° C., in2×SSC containing 0.1% w/v SDS (moderate stringency) andautoradiographed. Following development of the autoradiographs, stripswere re-washed at a high stringency (0.1×SSC, 0.1% w/v SDS at 65° C.)and re-autoradiographed.

For Northern Blots For Probes M1, M1AUS and B1A (SEQ ID NOS: 1, 5 and 2Respectively)

The Northern blot of mRNA from 11, 15 and 23 day-old Haemonchuscontortus was probed first with the M1 insert. The filter wasprehybridised for 2 hours at 42° C. in 2×SSC (where 20×SSC=3 M NaCl, 0.3M sodium citrate, pH 7.2) containing 5×Denhardt's (0.1% w/v Ficoll 400(Pharmacia), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albuminFraction V (Sigma Chemical Corp)), 0.5% SDS (sodium dodecylsulphate),10% dextran sulphate, 0.1 mg/ml salmon testes DNA and 50% de-ionisedformamide. The hybridisation to the probe was performed in the samebuffer overnight at 42° C. The filters were washed twice for 30 minutesin 2×SSC containing 0.5% SDS and 50% formamide, twice for 30 minutes in2×SSC containing 0.5% SDS and twice for 30 minutes in 2×SSC. The firstwash was at 42° C. and all remaining washes were at 37° C. Afterautoradiography the blot was stripped by washing in boiling 0.1% SDS andre-autoradiographed to ensure removal of the probe. The same blot wasthen probed with the M1AUS insert, washed and autoradiographed. The blotwas again stripped and checked and when clear was then probed with theB1A insert. After stripping again the blot was then probed as describedbelow.

For Probes AustB1, 2.5 kb and 3.5 kb PCR Products (SEQ ID NOs: 6, 7 and8)

The Northern blot was hybridised with the AustB1 insert using theconditions of moderate stringency as described for Southern blothybridisation. After autoradiography the blot was stripped with boiling0.1% SDS according to the membrane manufacturer's instructions(Amersham), then probed with the 2.5 kb PCR insert (clone 2), strippedagain and probed with the 3.5 kb PCR (clone 2) insert.

Digestion of H110D and Assays of Enzyme Activity Preparation of H110D

Native H110D (H110DN) was prepared according to the methods described inWO88/00835 and WO90/11086.

Preparation of an Elastase Fragment (H11S) of H110D

Adult Haemonchus were homogenized in 10 volumes of ice cold PBS/0.02%sodium azide and then centrifuged for 20 minutes at 13000 rpm. Thepellet was resuspended in 10 volumes of PBS/azide, rehomogenised and thecentrifugation repeated. Following resuspension in 50 mM MOPS buffer pH7.4 (the volume for suspension is 1 ml for each 0.17 g of worms) andpre-warming at 37° C. for 30 minutes, the pellet material was digestedwith elastase (800 μl/20 ml of suspension; 1 mg/ml fresh stock solutionmade up in 1 mM HCl/2 mM Ca²⁺) for one hour. The digestion was stoppedby the addition of 3,4 dichloroisocoumarin (300 μl of stock 10 mM inDMSO/200 ml of digest). The mixture was centrifuged at 13000 rpm for 20minutes and the pelleted material retained. The supernatant wasultracentrifuged at 100000 g for 1 hour 20 minutes. The resultantsupernatant liquid was applied to a ConA column and the bindingfractions obtained. For analysis, this fraction was run on anSDS-polyacrylamide gel and electrophoretically transferred topolyvinylidene difluoride membrane (Immobilon-P, Millipore), lightlystained with Coomassie blue, the 105 kd band excised and analysed in agas phase amino acid sequenator. For vaccination studies, the ConAbinding fractions were further purified by concentrating and applying toa Superose 12 (Pharmacia) gel filtration column and collecting thosefractions containing aminopeptidase activity.

Thermolysin Digestion of H110D

The H110D doublet was purified by electroelution from a preparativescale 8% SDS-polyacrylamide gel to give H110DE, as described inWO90/11086 and by electroelution of a dimeric form, running at just over200 kd (which yielded the characteristic doublet at 110 kd when re-runon SDS-PAGE) to give H110DE. Solutions of H110DE were concentrated to200 g, calcium chloride was added to 5 mM and the mixture warmed to 37°C. A freshly prepared solution of 1 mg/ml Thermolysin (in 1 mM HCl, 2 mMCaCl₂) was added in a ratio of 0.1 μg Thermolysin per μg H110DE. Themixture was incubated at 37° C. for 120 minutes and the reaction thenstopped by addition of 5 μl of 0.5M EDTA.

The protein fragments were separated by 15% SDS-polyacrylamide gelelectrophoresis and electrophoretically transferred to polyvinylidenedifluoride membrane (Immobilon-P, Millipore). Following staining withCoomassie blue the most intense discrete bands were excised and analysedin a gas phase amino acid sequenator.

Preparation of an H110D Fraction (H11A) Enriched for Aminopeptidase AActivity

The pelleted material obtained after elastase treatment bycentrifugation at 17000 g for 20 min. (see above) was resuspended in PBSat 4° C. and repelleted by centrifugation then resuspended in 1% Tweenin PBS/azide and left (with stirring) for 1 hour. The suspension wascentrifuged at 17.000 g for 20 minutes and the supernatant removed. Thepellet was repeatedly extracted with 1% Thesit in PBS/azide. Thesupernatants after centrifugation at 17,000 g for 20 minutes werecombined and ultracentrifuged for 1 hour 20 minutes at 100,000 g. Thesupernatant was applied to a ConA affinity column (Affigel, Biorad) andthe bound material eluted and further fractionated by ion exchangechromatography on a MonoQ column.

Assays of Enzyme Activities

α-amino acylpeptide hydrolase (microsomal) aminopeptidase activities inH110D preparations were characterised by assays, in solution, usingL-leucine, methionine, phenylalanine, α-glutamic acid and lysinep-nitroanilides (pNA). All the amino acid p-nitroanilide substrates(except α-glutamic acid-) were obtained from Sigma, Poole, Dorset UK,α-glutamic acid was from Fluka, Dorset, UK. Single hydrophobic (leucine-or phenyalanine-) and charged (α-glutamic acid-) amino acid pNA known tobe substrates for mammalian aminopeptidase-M (ApM) and -A (ApA)respectively, were chosen to measure the effect of enzyme inhibitors andserum inhibition on H110D aminopeptidase activities.

(a) Microplate Assay

Micro-ELISA plate (Dynatech Immulon 1, Virginia, USA) wells were eachfilled with 250 μl of either 50 mM HEPES or MOPS pH 7 plus 1-10 μl ofthe fraction to be assayed. The plates were then pre-incubated at 37° C.for 10 minutes prior to the addition of 10 μl of 25 mM amino acidp-nitroanilide substrate per well. The time zero optical density (OD) at405 nm was then measured using an ELISA plate reader and the plates werethen incubated at 37° C. for 15-30 minutes. The final OD reading wasthen taken as before and the OD change per minute per milligram ofprotein calculated.

(b) Inhibitor Sensitivity

The method used for the enzyme assay was as described in (a) aboveexcept that the inhibitors were added to the 250 μl of buffer plus 10 μlof 1 mg/ml ConA H110D and pre-incubated for 10 minutes at 37° C. priorto addition of the substrates (leucine-pNA or α-glutamic acid-pNA). Thepercentage inhibition was calculated as follows

x−y/x×100=percentage inhibition.

Where x=the ΔOD/min of the enzyme with no inhibitor added and y=ΔOD/minof the enzyme plus inhibitor. Nine compounds with differing enzyme classinhibition were tested individually: Amastatin, Bestatin(metalloprotease, aminopeptidase), 1,10 phenanthroline, EDTA(metalloprotease), phosphoramidon (metalloprotease, thermolysin,collagenase), Aprotinin (serine protease), Pepstatin (asparticprotease), PMSF (serine and cysteine protease) and E64 (cysteineprotease). Amastatin, bestatin, 1,10 phenanthroline, PMSF and pepstatinwere obtained from Sigma, Dorset, UK. All the other inhibitors wereobtained from Boehringer-Mannheim. Each inhibitor was used at twoconcentrations equal to or greater than the maximum concentrationsrecommended by Boehringer-Mannheim.

(c) Assay of Antiserum Inhibition of Enzyme

The assay was as described in (a) above except that two micro-ELISAplates were set up. In one plate 10 μl of ConA H110D (1 mg/ml) plus 10μl of antiserum from each sheep per well were pre-incubated at 37° C.for 15 minutes. The second plate, with 250 μl of HEPES/bicarbonatebuffer plus 10 μl of either phenylalanine-pNA or α-glutamic acid-pNAsubstrate per well was also pre-incubated at 37° C. for 15 minutes. TheConA H110D-serum mixtures were then added to the buffer-substratemixtures in the second plate with a multi-pipette. The ΔOD/min wasdetermined. For the purposes of the correlations, percentage inhibitionwas calculated using the formula in (b) above here x represents the meanΔOD/min for the wells which contained enzyme plus serum from negativecontrol sheep (vaccinated with horse spleen ferritin) and y=the ΔOD/minfor the wells which contained enzyme plus sera from individual sheepvaccinated with H110D. For the purposes of the correlations (FIG. 18),the percentage protection was calculated using the formula in (b) abovewhere x=either the average faecal egg output per gram or the averageworm burden of the controls, and y=either the faecal output per gramduring the experiment or the worm burden of the individual sheepvaccinated with H110D.

Localisation of Enzyme Activity by Histochemistry

Aminopeptidase activity was demonstrated on 10 μm cryostat sections ofadult Haemonchus contortus using L-leucine 4-methoxy-β-naphthylamide andL-glutamic α-4-methoxy-β-naphthylamide as substrates by the methods ofNachlas et al. (1957), Nakane et al. (1974) and Lojda et al. (1980).

Expression of Recombinant H110D in the Eukaryotic Baculovirus-InsectCell System Construction of Expression Plasmids

The 3.5K PCR fragments described above, were generated by amplificationbetween an oligo dT adaptor (which contained a SalI site) and oligo 872,which represents base no.'s 30-49 in the M1AUS sequence, and were clonedinto the pT7Blue vector. Clone pT73.5-2 is oriented with the vectorpolylinker BamHI site at its 5′ end and the HindIII, XbaI and SalI sitesat the 3′ end. The sequence at the 5′ end is:

    BamHI 5′ GG ATC CGA  *!-----oligo 872-----------!--3.5K----- TTG CTGAAT CTA ACT CCA ATC C .......3′     Leu Asn Leu Thr Pro Ile .........The asterisk indicates the 3′ dT/dA overhang used for cloning in the pT7Blue Vector.

The 3.5 PCR clone 2 was digested with HindIII (single site in the vectorpolylinker sequence at the 3′ end of the 3.5K gene) and the ends filledin with deoxynucleotides using DNA polymerase I (Klenow fragment),according to Maniatis et al (1982). A BamHI linker (5′CGGATCCG 3′, NewEngland Biolabs Catalog no. 1021) was ligated to the blunt ends, andclones with an extra BamHI site, allowing a full-length H110D genesequence to be excised as a BamHI fragment, selected. The BamHI fragmentwas isolated by electrophoresis on a 0.6% w/v agarose gel intris-acetate buffer, followed by purification using GENECLEAN (BIO101);this procedure was carried out twice. The purified fragment was thenligated to BamHI-cut, phosphatased pBlueBac II (Invitrogen Corp.), andclones carrying the fragment in the correct orientation (ie. with the 5′end of 3.5 PCR clone 2 placed under control of the baculoviruspolyhedrin promoter) determined by digestion with NheI and XbaI. Theresultant plasmid was partially digested with BamHI and the ends filledin as described above. An NcoI linker containing an ATG (5′CCCATGGG 3′;New England Biolabs Catalog no. 1040) was added and the mixture ligated.Clones which had the linker ligated at the 5′ end BamHI site of 3.5 PCRclone 2 rather than the 3′ site, were determined by digestion with NcoI.The resultant plasmid, designated pBB3.5-2(N), is depicteddiagrammatically in FIG. 19.

This construction results in the insertion of an in-frame ATG at the 5′end of the 3.5 PCR clone 2 insert, to initiate translation. The sequencesurrounding this initiating ATG is:

  BamHI--NcoI link-BamHI----*!-----oligo 872-----------!-- 5′GGATCCCCATG GGG ATC CGA TTG CTG AAT CTA ACT CCA ATC C..            Met Gly IleArg Leu Leu Asn Leu Thr Pro Ile ...The expressed protein will be missing amino acids 2-9 of thecorresponding H110D sequence, and will have 3 amino acids of linkersequence immediately following the ATG.

Generation of Recombinant Baculovirus Containing H110D Sequences

The plasmid pBB3.5-2(N) was transfected into Spodoptera frugiperda (Sf9)cells (obtainable from Invitrogen Corp), using linear Autographicacalifornica nuclear polyhedrosis virus (ACNPV) DNA and cationicliposomes (Invitrogen Corp. transfection module), according to themanufacturer's instructions-. Cells were cultured in TC-100 medium(SIGMA) supplemented with foetal calf serum (CSL Ltd; heat-inactivatedat 56° C. for 45 minutes) and antibiotics (penicillin/streptomycin,gentamycin; CSL Ltd). A control transfection, using a pBB3.5-2 (N)plasmid with the ATG inserted at the 3′ end of the 3.5 PCR clone 2sequence, was also carried out. Recombinant plaques were selected on thebasis that the pBlueBac II vector also encodes E. coli β-galactosidase(β-gal), by including X-gal in the agarose overlay at the recommendedlevel. A selection of blue plaques were picked and subjected to twofurther rounds of plaque purification, after which time infectedmonolayers showed no evidence of contaminating wild-type virus (whichwould be evidenced by the presence of nuclear polyhedra). Purifiedviruses were designated 3.5-2-P2A, -P3A and -P4A, and were amplified bytwo sequential infections of Sf9 cells before use. A plaque purifiedfrom the control transfection was designated 3.5-2-rev.

Assessment of H110D Expression in Insect Cells Infected with RecombinantBaculovirus

Monolayers of Sf9 cells (1×10⁶ cells in 25 cm² bottles) were infectedwith the 3.5-2 viruses, with wild-type (wt) virus, with a control virusexpressing β-gal, or were not infected. After four days growth at 26°C., monolayers were detached by gentle shaking, the cells recovered bycentrifugation (2000 rpm, 10 minutes), and the cell pellets disrupted bythree cycles of freeze-thawing. The lysates were resuspended in 500 μlPBS, and 25 μl aliquots assayed for ApM activity by the micro-wellassay.

15 μl aliquots (3×10⁴ cell equivalents) of the above lysates wereelectrophoresed on denaturing 7.5% SDS-polyacrylamide gels. One gel wasthen stained with Coomassie blue to assess levels of expression. Theother gel was Western blotted, and the blot probed with anti-H110DN (asdescribed earlier).

Results Analysis of Immunopositive Clones Analysis of AntibodiesAffinity Purified on Clone M1

Affinity-purified antibodies specific for each of the 5antibody-positive clones were prepared and used to probe a Western blotof H110D-enriched extract. As shown in FIG. 8, all 5 clones appeared torecognise the H110D doublet. However, the reaction with clone M1 gavethe strongest signal (FIG. 8 d) compared to the λgt11 negative controlblot (FIG. 8 e). This clone was therefore investigated further.

Northern Blot Analysis With Clone M1

Northern blot analysis of Haemonchus contortus mRNA probed with the M1insert is shown in FIG. 9. A single mRNA band was recognised, atapproximately 3.5 kb. This is of sufficient size to code for a proteinof about 110 kd.

Sequence Analysis of Clone M1

Analysis of restriction digests of the DNA with EcoRl showed the M1insert to be approximately 300 bp. The DNA sequence or the M1 fragmentwas determined (SEQ ID NO: 1, and is shown in FIG. 3. The fragmentcomprises 295 bp with an open reading frame starting at base number 3.

Northern Blot Analysis with Clone B1A

Northern blot analysis of Haemonchus contortus mRNA probed with the B1Ainsert is shown in FIG. 9 c. As for M1, a single mRNA band wasrecognised, at approximately 3.5 kb.

Sequencing of Clones B1A and B2

Clones of B1A were sequenced (SEQ ID NOS: 2 and 3) and the full sequence(SEQ ID NO: 2) is shown aligned to H11-1 (SEQ ID NO: 19) and AustB1 (SEQID NO: 6) in FIG. 5. The insert is 484 bp and has a full ORF from thefirst base. The 3 fragments of B2 resulting from digestion with EcoRlwere sequenced and the complete sequence for B2 (SEQ ID NO: 4) is 581bp. It is shown aligned with H11-2 in FIG. 4. The sequence has an ORFfrom position 3 to 213 bp, the stop codon and untranslated regionmatching that of the 2.5 kb PCR product sequence (SEQ ID NO: 7).

Expression of M1 and B1A in E. coli

When subcloned into a GST expression vector, clones were obtained whichexpressed fusion proteins of 38-40 kd for M1 and of 45 kd for B1A. Theseagree with the predicted sizes for these inserts, allowing for themolecular weight of glutathione-S-transferase. Both fusion proteinsreacted very strongly on Western blots with affinity-purified antibodiesto H110DE (FIG. 11). The fusion proteins were expressed as insolubleinclusion bodies.

Antibody Responses in Sheep Vaccinated with M1-GST and B1A-GST FusionProteins

Antisera from sheep injected with the fusion proteins were tested byWestern blotting against H110D preparations. Both GST-M1 and GST-B1Araised antibodies which specifically recognised the H110D doublet (FIG.12). Sera from negative control sheep did not recognise the H110Ddoublet.

Isolation and Characterisation of Clones Selected by Hybridisation withM1 or B1A Insert DNA

The confirmed positive clone hybridising to the M1 probe was designatedM1AUS (SEQ ID NO: 5), and the clone hybridising to B1A was designatedAustB1 (SEQ ID NO: 6). Restriction digestion of purified plasmid DNAswith EcoRI indicated an insert size of about 900 bp for M1AUS and ofabout 1.6 Kb for AustB1. As shown in FIG. 9 b) and 9d), on Northernblots, M1AUS and AustB1 hybridised to the same-sized mRNA (about 3.5 kb)as did M1 and B1A.

Sequence Analysis of M1AUS

Full sequencing of the M1AUS fragment was carried out using syntheticoligonucleotides to “walk” along the DNA from either end. Analysis ofthe sequence obtained revealed that the M1AUS insert was 948 bp, asshown in FIG. 3. The sequence (SEQ ID NO: 5) begins with an ATG (whichcodes for methionine) and has an open reading frame (ORF) over the wholeof its length. The sequence is 19 base pairs longer than the M1 sequenceat the 5′ end, and 634 bp longer at the 3′ end. The sequence common tothe two clones (bases 20 to 314) were identical except for twonucleotide differences in a third codon position. Comparison of allpossible reading frames to various databases showed that the readingframe starting with the ATG at base number one shared homology with themembers of a family of microsomal aminopeptidases.

Sequence of AustB1

Full sequencing of the AustB1 fragment was carried out using syntheticoligonucleotides to “walk” along the DNA from either end. The DNAsequence (SEQ ID NO: 6) is shown in FIG. 5. The clone is 1689 bp longand has an ORF from residue 2. This sequence forms part of H11-1 asshown in FIG. 1. The amino acid translation of this sequence showed thezinc binding site motif characteristic of aminopeptidases.

PCR Amplification of the cDNA of the H110D mRNAs PCR using M1 AUSPrimers

cDNA was synthesized from Haemonchus contortus mRNA using as primeroligo-dT containing an adaptor sequence to facilitate subsequent cloningand manipulation of the DNA. This cDNA was then used to amplify theM1AUS sequence by PCR, using as the 5′ end primer a syntheticoligonucleotide based on positions 865-885. A PCR fragment of about 2.5Kb was amplified. This is approximately the expected size of thefragment, based on the known size of the mRNA and on mammalianaminopeptidase cDNA sequences.

A second set of PCR reactions was performed using a primer near the 5′end of M1AUS (bases 30-49). Four bands were detected on an agarose gel.The largest of these, at 3.5 kb, corresponds to the predicted size forthe PCR product.

Cloning and Sequencing of 2.5 kb and 3.5 kb PCR Products from M1AUSPrimers

The 2.5 kb and 3.5 kb PCR products were cloned and designated 2.5PCR(SEQ ID NO: 7) and 3.5PCR (SEQ ID NOS: 8, 14 and 15 for clone numbers 2,10 and 19 respectively). On Northern blots 2.5PCR and 3.5PCR (clone 2.3.5PCR-2) hybridised with mRNA of about 3.5 kb (FIGS. 9 e, f) in thesame pattern (with respect to age of Haemonchus used to obtain the mRNA)as M1, B1A, M1AUS and AustB1.

Full sequencing of clones was carried out by ‘oligonucleotide walking’,As shown in FIG. 1, the sequence for the 2.5 kb product (SEQ ID NO: 7)is part of H11-2 (SEQ ID NO: 20) and the sequence for the 3.5 kb product(SEQ ID NO: 8) is the major part of H11-3 (SEQ ID NO: 21). The aminoacid translations of both these sequences (shown in FIG. 6) contain thezinc binding motif His Glu Xaa Xaa His Xaa Trp (HEXXHXW) characteristicof microsomal aminopeptidases.

Sequencing of 5′ End PCR Clones

cDNA was synthesised using a primer matching a conserved sequence incDNA clone AustB1, 2.5PCR and 3.5PCR (SEQ ID NOS: 6, 7 and 8) whichhybridises with the mRNA for these sequences about 1.3 Kb from the 5′end. The cDNAs were C-tailed at the 5′ end and then PCR reactionscarried out with a universal Anchor (A) primer for the 5′ end and threeprimers specific for each of the sequences AUSTB1, 2.5PCR and3.5PCR-Clone 2 (SEQ ID NOS: 6, 7, 8) for the 3′ end. The reactions eachgave a product of the predicted size, just under 1.3 kb: 1301 bp (SEQ IDNO: 9), 1280 bp (SEQ ID NO: 10) and 1292 (SEQ ID NO: 11) respectively.All three sequences have an untranslated region at the 5′ end (FIG. 2).All begin with the same 22 bp sequence (5′ GGTTTAATTACCCAAGTTTGAG 3′)which is known as the Spliced Leader Sequence 1 (SL1) and is present inthe untranslated 5′ region of a wide variety of nematodes Huang et al.,1990. In SEQ ID NOS: 9 and 10, the SL1 sequence is immediately beforethe initiating ATG. In SEQ ID NO: 11 there are 13 bp between the SL1 andthe initiating ATG. All three sequences have full ORFs.

Sequencing of AustB1 3′ End PCR Clone

Using a specific primer matching positions 1414-1438 in Aust B1 (SEQ IDNO: 6), the PCR product gave a band as predicted of about 1.3 kb.Sequencing of the cloned band yielded the sequences SEQ ID NOS: 12 and13. They gave an ORF from 1-615 bp and a substantial untranslatedregion.

Sequence Analysis of Cloned PCR Products

Composites of the sequences described above, designated H11-1, H11-2 andH11-3, are shown in FIG. 2. The amino acid sequences predicted fromthese are shown in FIG. 6 a. The validity of the predicted translationsof the DNA sequences presented is substantially confirmed by the matcheswith amino acid sequences determined by Edman degradation from CNBr andproteolytic cleavage fragments (FIG. 7). Thus 27 residues of the 29residue N-terminal sequence of H11S (SEQ ID NO: 16) match H11-2 fromresidues 61-90 (FIG. 7 b). The matches of valine (V) at position 78 andglycine (G) at position 90 are characteristic of H11-2 since H11-3 hasasparagine (N) at position 90 and H11-1 has leucine (L) at position 86(which corresponds to position 78 in H11-2). Two residues of the H11SN-terminus amino acid sequence (SEQ ID NO: 16) do not match any of thethree H110D sequences presented here. To be particularly noted are theexact matches of the very similar, but distinctive sequences Pep A and B(previously described in WO90/11086) with H11-2 and H11-1 respectivelyin the region of residues 540-555. Similarly in the 450-470 residueregion, Pep D is an exact match for H11-2, while the similar butdistinct Pep E matches more closely H11-3.

By way of example the translated amino acid sequence of one of thefull-length sequences (H11-3) is compared in FIG. 6 b with two sequencesfor mammalian microsomal aminopeptidases. The homology of the H110Dtranslation with these aminopeptidases is shown by boxing identicalamino acids. A characteristic motif of microsomal aminopeptidases is theamino acid sequence HEXXHXW, which functions as the zinc binding site(Jongeneel et al., 1989; Vallee et al., 1990); this is shown byasterisks in FIG. 6. This sequence, which is shown to be present in thetranslations of H11-1, H11-2 and H11-3, is conserved in all themicrosomal aminopeptidases. Other features common to the mammalian andHaemonchus microsomal aminopeptidases are the presence of acomparatively short intracellular region, a single transmembranesequence adjacent to the N-terminus and several potential glycosylationsites. The levels of homology (similarities of 52-55% and identities of30-32%) of H11-1, -2 and -3 to mammalian microsomal aminopeptidases areshown in Table 2.

Southern Blot Analyses

The results of H. contortus genomic DNA Southern blots probed withvarious H110D cDNA clones and PCR products are shown in FIG. 10. Allprobes show multiple bands of hybridisation; this is typical of amultigene family. As expected, B1A and AustB1 showed similarhybridisation patterns to each other, as did M1AUS and 3.5 kbPCR.However, these patterns were noticeably different from each other andfrom that seen with the 2.5 KbPCR probe, even under conditions ofmoderate stringency (FIG. 10A), reflecting the differing levels ofhomology between these three cDNAs.

Demonstration of Aminopeptidase Activities Associated With H110D

Microsomal aminopeptidase activity was found to associate with thosefractions containing H110D, that is the supernatants fromultracentrifugation of Thesit extracts, ConA binding fraction (ConAH110D) and the fractions containing H110D obtained by ion exchangechromatography on a MonoQ column (Table 3). The specific activities withall substrates tested increased as the purity of the H110D increased.

TABLE 3 ENZYME ACTIVITIES OF FRACTIONS FROM A TYPICAL H110D PREPARATIONSPECIFIC ENZYME ACTIVITIES (O.D./minute/mg protein) Phenyl- α- alanine-Leucine- Lysine- Glutamic Methionine- FRACTION pNA pNA pNA acid-pNA pNAPhosphate 0.20 0.08 0.12 0.01 0.16 buffered saline (PBS) 1% Tween 0.150.15 0.09 0.01 0.09 20/PBS 1% Thesit/PBS 1.94 1.25 0.57 0.54 1.91 ConAH110D 4.08 3.01 1.43 2.09 3.84 CamQ H110D 6.55 5.01 3.01 3.90 6.41

Effects of Inhibitors of Mammalian Aminopeptidases on H110DAminopeptidase Activities

Addition of the inhibitor bestatin (which inhibits mammalian microsomalaminopeptidase) to ConA H110D at the concentration recommended by thesupplier (Boehringer Mannheim) reduced the activity againstleucine-p-nitroanilide by approximately 70%. A series of experimentswere performed to test inhibition of activity by a range of proteaseinhibitors. Those inhibitors which were not specific formetalloproteases or aminopeptidases had no inhibitory effects on thereaction rate. Inhibitors that are known to affect metalloproteases oraminopeptidases did have an effect on reaction rates, as shown in Table4. The most effective inhibitor was 5 mM phenanthroline.

TABLE 4 Inhibition of H110D aminopeptidase activities using variousprotease inhibitors PERCENTAGE PERCENTAGE INHIBITION INHIBITIONα-glutamic acid- Leucine-p- CONCEN- p-nitroanilide nitroanilideINHIBITOR TRATION substrate¹ substrate² Amastatin 10 μM 0 61 50 μM 0 66Bestatin 50 μM 23 63 100 μM 35 68 EDTA 1 mM 17 8 10 mM 21 16 1,10phenanthroline 1 mM 78 78 10 mM 85 87 Phosphoramidon 10 μM 1 0.8 50 μM 17 ¹A mammalian aminopeptidase-A substrate. ²A mammalian aminopeptidase-Msubstrate.

Sub-Fractionation of H110D

The distribution of activities associated with fractions from ionexchange chromatography of ConAH110D on MonoQ are shown in FIG. 13 a andSDS-PAGE of the fractions in FIG. 13 b.

Further, enzymatic activity was associated with sub-fractions of H110Dseparated by re-cycling free flow isoelectric focussing (FIGS. 14 and15). At lower pI values (pH 4.5) these sub-fractions contain only thelarger of the bands which make up the H110D doublet seen on SDS-PAGE andat higher values (pH 6.5) they contain only the smaller of bands whichmake up the H110D doublet. Intermediate fractions contain both thesebands. The smaller band may also be obtained as a separate fraction byion exchange chromatography on MonoQ-using the Pharmacia SMART®apparatus. All these sub-fractions bind sheep antibodies to H110Daffinity purified on the protein expressed by the λgt11 clone M1 whereasantibodies eluted from λgt11 with no insert do not bind (FIG. 14 c). Allthe sub-fractions bind mouse monoclonal antibodies designated TS 3/19.7(FIG. 14 c) which also bind to the recombinant protein expressed byclone M1. All the sub-fractions show microsomal aminopeptidase activity(FIG. 15 c) although this activity is comparatively low in the fractionsobtained at the highest and lowest pIs. This lower activity may beattributed to lowered protein concentrations, effects of extremes of pHduring sub-fractionation or a requirement for the presence of bothlarger and smaller bands for maximal activity.

Vaccination with Separated Components of H110D

The separated upper and lower bands obtained by free flow isoelectricfocussing or by ion exchange chromatography induce the formation ofprotective antibodies when injected into sheep as exemplified in thefollowing experiment. Thirty sheep approximately six months old wereassigned to 5 groups of 6 so that each group was matched for range ofweights of sheep. Each animal was injected with a total of 150 μgprotein given in 3 equal doses as described in Munn et al. (1992) andTavernor et al. (1992a, b) over a period of 54 days. The animals ingroup L were injected with the lower (smaller) band of the H110 Doublet,those in group U with the upper band, U+L with recombined upper andlower bands, D with the two (unseparated) bands obtained by free-flowisoelectric focussing at intermediate pH values and as a control (groupC) horse spleen ferritin (an antigenic unrelated protein). The sheepwere challenged with 10,000 infective larvae three weeks after the thirdinjection and the experiment terminated 29-31 days post infection. Theoutcome of the experiment is summarised in FIG. 16. Injection of any ofthe sub-fractions reduced parasite egg output throughout the trial bysome 90% and reduced total worm numbers by 63-84%, all showing asignificant difference (p<0.05) to the controls using non-parametricstatistical analyses. Reductions (70-88%) in the numbers of female wormswere greater than the reductions in the numbers of male worms, and(except for the reduction in male worm numbers in the sheep injectedwith the recombined upper and lower bands where p<0.07) for both sexesthe reductions were significant (p<0.05).

Vaccination with H11S and H11A

A truncated, water-soluble form of H110D (H11S; which retains itsenzymic activity) may be obtained from the native molecule by treatmentwith elastase. This form was found to contain predominantly ApM-likeenzyme activity and a Thesit extract of the elastase digested pellet(H11A) was enriched for ApA-like activity (see Table 5).

TABLE 5 Ratio Aminopeptidase-M:Aminopeptidase-A (leucine-pNA)(α-glutamic acid-pNA) H110D 1.44:1 H11S 26.0:1 H11A 0.48:1The following experiment shows that vaccination of sheep with eitherH11S or H11A gives protection against Haemonchus challenge or infection.Eighteen sheep approximately eight months old were assigned to 3 groupsof 6 so that each group was matched for range of weights of sheep. Eachanimal was injected with a total of 100 μg protein given in 2 equaldoses as described in Munn et al. (1992) and Tavernor et al. (1992a, b)over a period of 23 days. The animals in group A were injected withH11A, those in group S with H11S and those in group C with horse spleenferritin (an antigenically unrelated protein) as a negative control. Thesheep were then challenged with 10,000 infective larvae 25 days afterthe second injection and the experiment terminated at 34-36 days postinfection. The outcome of the experiment is summarised in FIG. 17.Injection of H11S reduced parasite egg output throughout the trial by89% and reduced total worm numbers by 76%. Injection of H11A reducedparasite egg output throughout the trial by 98% and reduced total wormnumbers by 84%. These showed a significant difference (p<0.05) from thecontrols using non-parametric statistical analyses.

Inhibition of H110D Aminopeptidase Activities by Antibodies

Solutions containing H110D were incubated with sera from individualsheep injected with fractions containing H110D or from control sheep.The solutions were then assayed for aminopeptidase activities usingphenylalanine and α-glutamic acid pNAs as substrates. The degree ofinhibition of activity (maximally 80%) correlated with the level ofprotection shown by the individual sheep from which the sera wereobtained (see FIG. 18).

Localisation of Enzyme Activity

Frozen sections of adult Haemonchus contortus were examined foraminopeptidase activity. As shown in FIG. 19, aminopeptidase enzymeactivities are localised to the luminal surface of the intestine. H110Dprotein is also specifically found in this location.

Expression of H110D (3.5 PCR Clone 2) Using the EukaryoticBaculovirus-Insect Cell System Expression of Aminopeptidase Activity inInsect Cells

Infected cells were harvested and assayed for aminopeptidase activitiesusing phe-, leu-, met- and lys-pNA as substrates. The assay wascomplicated by the observation that the insect cells possess anaminopeptidase activity with a marked preference for lys-linked amidebonds. However, cell extracts containing the expressed H110Dadditionally cleaved leu-, met- and phe-pNA in that order of preference.

Molecular Weight and Immunoreactivity of the Expressed H110D (3.5 PCRclone 2) Protein

Samples of infected or control cell extracts were electrophoresed on a7.5% SDS-polyacrylamide gel, which was then stained with Coomassie Blue.The 3.5-2-P3A and 3.5-2-P4A infected cell lysates both had a band at thesame size as H110D, 110 kd, which migrated directly beneath theco-expressed β-gal, which has a molecular weight of 120 kd. This 110 kdband was not present in any of the negative control lysates. (It wasalso absent from the P2A lysate, which did not express enzyme activity).

A duplicate gel was Western blotted and probed with anti-H110DN (FIG.21). A very strong, specific positive immunoreaction was obtained to the110 kd band expressed by 3.5-2-P3A and 3.5-2-P4A, and to the nativeH110D doublet in a control track containing ConA H110D, while noreaction was seen in any of the negative control tracks.

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1. A synthetic polypeptide comprising an amino acid sequence encoded bya nucleotide sequence which encodes a helminth aminopeptidase or anantigenic portion thereof, said nucleic acid selected from the groupconsisting of i) any sequence of SEQ ID NOs:1-15 or 19-21, ii) asequence fully complementary to said sequence of (i), iii) a portion ofsaid sequence of (i) wherein said portion of said sequence encodes anantigenic peptide of said helminth aminopeptidase, and (iv) a sequencewhich hybridizes with said sequence of (i) or (ii) under conditions of 2times SSC, 65 C (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2)and which has at least 60% identity with said sequence of (i), otherthan a synthetic polypeptide corresponding to the protein doublet H110Dor a synthetic polypeptide selected from: (SEQ ID NO: 25) (a) Met GlyTyr Pro Val Val Lys Val Glu Glu Phe, (SEQ ID NO: 26) (b) Met Gly Phe ProVal Leu Thr Val Glu Ser, (SEQ ID NO: 27) (c) Met Gly/Phe Asn Phe Lys IleGlu/Val Thr/Glu Ala Gly, (SEQ ID NO: 28) (d) Met Lys Pro/Glu Thr/Val LysAsp/Ala Thr/Lys Leu - Ile Thr, (SEQ ID NO: 29) (e) Met Leu Ala Leu AspTyr His Ser - Phe Val, (SEQ ID NO: 30) (f) Met Leu Ala Glu/Tyr AspGln/Ala Glu Asp Val, (SEQ ID NO: 31) (g) Met Gly Phe Pro Leu Val Thr ValGlu Ala Phe Tyr, (SEQ ID NO: 32) (h) Met Lys Thr Pro Glu Phe Ala Val/LeuGln Ala Phe/Thr Ala Thr Ser/Gly Phe Pro, (SEQ ID NO: 33) (i) Lys His/TyrAsn/Val Ser Pro Ala Ala Glu Asn/ Leu Leu Asn/Gly, (SEQ ID NO: 34) (j)Lys - Thr Ser Val Ala Glu Ala Phe Asn, (SEQ ID NO: 35) (k) Lys Ala AlaGlu Val Ala Glu Ala Phe Asp - Ile - - - Lys Gly, (SEQ ID NO: 36) (l) LysAla Val Glu Val/Pro Ala Glu Ala Phe Asp Asp Ile Thr? Tyr - - Gly ProSer, (SEQ ID NO: 37) (m) Lys - Glu Glu Thr Glu Ile Phe Asn Met, (SEQ IDNO: 38) (n) Lys - - - Pro Phe Asn/Asp Ile Glu Ala Leu, (SEQ ID NO: 39)(o) Asp Gln Ala Phe Ser Thr Asp Ala Lys, (SEQ ID NO: 40) (p) Met Gly TyrPro Val Val Lys Val Glu Glu Phe - Ala Thr Ala Leu, (SEQ ID NO: 41) (q)Met Gly Phe Pro Val Leu Thr Val Glu Ser - Tyr? - Thr, (SEQ ID NO: 42)(r) Met Glu/Phe Asn Phe Leu Ile Glu/Val Thr/Glu Ala Gly - Ile Thr, (SEQID NO: 43) (s) Met Gly Phe Leu Val Thr Val Glu Ala Phe Tyr - Thr Ser,(SEQ ID NO: 44) (t) Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala Phe/ThrAla Thr Ser/Gly Phe Pro (SEQ ID NO: 45) (u) Met Lys Pro/Glu Thr/Val LeuAsp/Ala Thr/Lys Leu - Ile Thr - Gly, (SEQ ID NO: 46) (v) Met Leu Ala LeuAsp Tyr His Ser - Phe Val Gly?, (SEQ ID NO: 47) (w) Met Leu Ala Glu/TyrAsp Gln/Ala Glu Asp Val, (SEQ ID NO: 48) (x) Lys His/Tyr Asn/Val Ser ProAla Ala Glu Asn/ Leu Leu Asn/Gly, (SEQ ID NO: 49) (y) Lys - Thr Ser ValAla Glu Ala Phe Asn, (SEQ ID NO: 50) (z) Lys Ala Ala Glu Val Ala Glu AlaPhe Asp - lIe - - - Lys Gly, (SEQ ID NO: 51) (aa) Lys Ala Val GluVal/Pro Ala Glu Ala Phe Asp Asp Ile Thr? Tyr - - Gly Pro Ser, (SEQ IDNO: 52) (bb) Lys - Glu Gln Thr Glu Ile Phe Asn Met, (SEQ ID NO: 53) (cc)Lys - - - Pro Phe Asn/Asp Ile Glu Ala Leu and (SEQ ID NO: 54) (dd) AspGln Ala Phe Ser Thr Asp Ala Lys.


2. A synthetic polypeptide comprising an amino acid sequence encoded bya nucleotide sequence which encodes a polypeptide which raisesprotective antibodies against helminth parasites, which nucleotidesequence incorporates at least one aminopeptidase-encoding sequence,selected from the group consisting of i) any sequence of SEQ ID NOS:1-15 or 19-21, ii) a sequence fully complementary to said sequence of(i), iii) a portion of said sequence of (i), and iv) a sequence whichhybridizes with said sequence of (i) or (ii) under conditions of 2 timesSSC, 65 C (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) andwhich has at least 60% identity with said sequence of (i), other than asynthetic polypeptide corresponding to the protein doublet H110D or asynthetic polypeptide as defined in (a) to (dd) in claim
 1. 3. Asynthetic polypeptide comprising an amino acid sequence encoded by anucleotide sequence which corresponds to or which is complementary toone or more sequences selected from the group of sequences consisting ofi) M1(SEQ ID NO:1), ii) B1A (SEQ ID NO:2), iii) B1A-3′ (SEQ ID NO:3),iv) B2 (SEQ ID NO:4), v) M1AUS (SEQ ID NO:5), vi) AusB1 (SEQ ID NO:6),vii) 014-015 (2.5 PCR) (SEQ ID NO:7), viii) 014-872 (3.5 PCR clone 2)(SEQ ID NO:8), ix) A-648 (5′ END of B1) (SEQ ID NO:9), x) A-650 (5′-endof 2.5 PCR) (SEQ ID NO:10), xi) α-649 (5′ end of 3.5 PCR) (SEQ IDNO:11), xii) 014-178 (3′ end of AustB1 clone 2) (SEQ ID NO:12), xiii)014-178 (3′ end of AustB1 clones 3 and 6) (SEQ ID NO:13) xiv) 014-872(3.5 PCR clone 10) (SEQ ID NO:14), xv) 014-872 (3.5 PCR clone 19) (SEQID NO:15), xvi) H11-1 (SEQ ID NO:19), xvii) H11-2 (SEQ ID NO:20), xviii)H11-3 (SEQ ID NO:21), and xix) a sequence which hybridizes with any ofsaid sequences of (i)-(xviii) under conditions of 2 times SSC, 65 C(where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has atleast 60% identity with said sequence of (i)-(xviii), other than asynthetic polypeptide corresponding to the protein doublet H110D or asynthetic polypeptide as defined in (a) to (dd) in claim
 1. 4. Asynthetic polypeptide as claimed in claim 1, 2 or 3 substantially freefrom other Haemonchus contortus components.
 5. A synthetic polypeptideas claimed in claim 1, 2 or 3 in the form of a fusion polypeptide,comprising an additional polypeptide fused to said amino acid sequenceas defined in any one of claims 1 to
 3. 6. A synthetic polypeptide asclaimed in claim 1, 2 or 3 linked to an oligosaccharide having thestructure:


7. A synthetic polypeptide as claimed in claim 6 wherein saidoligosaccharide has the structure:


8. A synthetic polypeptide as claimed in claim 1, 2 or 3 wherein saidsynthetic polypeptide is a recombinant polypeptide.
 9. A syntheticpolypeptide as claimed in claim 1, 2 or 3 prepared by a methodcomprising the steps: a) culturing a prokaryotic or eukaryotic cellcontaining a nucleic acid molecule comprising at least one nucleotidesequence which encodes a helminth aminopeptidase or an antigenic portionthereof, said nucleic acid selected from the group consisting of i) anysequence of SEQ ID NOs:1-15 or 19-21, ii) a sequence fully complementaryto said sequence of (i), iii) a portion of said sequence of (i) whereinsaid portion of said sequence encodes an antigenic peptide of saidhelminth aminopeptidase, and (iv) a sequence which hybridizes with saidsequence of (i) or (ii) under conditions of 2 times SSC, 65 C (whereSSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least60% identity with said sequence of (i), under conditions whereby saidsynthetic polypeptide is expressed, and b) recovering said syntheticpolypeptide thus produced.
 10. A vaccine composition for stimulatingimmune responses against helminth parasites in a human or non-humananimal, comprising at least one synthetic polypeptide as defined inclaim 1, 2 or 3, or a virus or host cell having inserted therein anucleic acid molecule encoding said polypeptide, for stimulation of animmune response to polypeptides encoded by the inserted nucleic acidmolecule, together with a pharmaceutically acceptable carrier.
 11. Amethod of stimulating an immune response against helminth parasites in ahuman or non-human animal comprising administering to said animal avaccine composition as defined in claim 10.